Inhibition of Connective Tissue Growth Factor Expression in Adult Retinal Pigment Epithelial-19 Cells by Blocking Yes-Associated Protein/Transcriptional Coactivator with PDZ-Binding Motif Activity.

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)-ß2. 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-ß2 treatment significantly raised the expression of not just YAP and TAZ, but also CTGF in ARPE-19 cells. TGF-ß2 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-ß2 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.

Hypoxia Tumor Microenvironment Activates GLI2 through HIF-1α and TGF-ß2 to Promote Chemotherapy Resistance of Colorectal Cancer.

BACKGROUND: A majority of relapse cases have been reported in colorectal cancer patients due to cancer stem cell progenitors. The factors responsible for chemoresistance have yet to be discovered and investigated as CSCs have reported escaping from chemotherapy's killing action. OBJECTIVE: In this study, we have investigated the effects of HIF-1α and TGF-ß2 in hypoxia conditions on the expression of GLI2, which is a potential factor for causing chemoresistance. Material and Methods. Colorectal samples of treated patients were collected from the Hospital Biological Sample Library. Culture of patient-derived TSs and fibroblasts was performed. The collected patient samples and cells were used for immunohistochemistry, quantitative PCR, and western blotting studies which were performed. RESULTS: It was reported that HIF-1α (hypoxia-inducible factor) and TGF-ß2 secreted from cancer-associated fibroblasts (CAFs) synergistically work to express GLI2 in cancer stem cells. Hence, it increased the stemness as well as resistance to chemotherapy. CONCLUSION: The HIF-1α/TGF-ß2-mediated GLI2 signaling was responsible for causing chemoresistance in the hypoxia environment. High expressions of HIF1α/TGF-ß2/GLI2 cause the relapsing of colorectal cancer, thus making this a potential biomarker for identifying the relapse and resistance in patients. The study uncovers the mechanism involved in sternness and chemotherapy resistance which will help in targeted treatment.

Targeting transforming growth factor-ß2 by antisense oligodeoxynucleotide accelerates T cell-mediated tumor rejection in a humanized mouse model of triple-negative breast cancer.

Transforming growth factor-beta (TGF-ß) pathway mediates suppression of antitumor immunity and is associated with poor prognosis in triple-negative breast cancer (TNBC). In this study, we generated a humanized animal model by transplanting human peripheral blood mononuclear cells into immunodeficient mice followed by inoculation of MDA-MB-231 cells and subsequently analyzed the role of TGF-ß2 in the interaction between human T cells and human tumor cells. Following reconstitution of the human immune system, inhibition of TGF-ß signaling by TGF-ß2 antisense oligodeoxynucleotide (TASO) resulted in accelerated tumor growth inhibition. TGF-ß2 inhibition also resulted in downregulation of peripheral Foxp3 + regulatory T cells (Treg), whereas no effect was seen in the expression of CD8 + cytotoxic T cells. Analysis of the TASO-treated mice serum revealed elevated levels of human IFN-γ and reduced levels of human IL-10 and TGF-ß2. Moreover, TGF-ß2 inhibition resulted in increased CD8 + T cell infiltration, whereas the reduced infiltration of Tregs into the tumor partly resulted from decreased expression of CCL22. Decreased intratumoral Tregs facilitated the activation of cytotoxic T cells, associated with increased granzyme B expression. These results indicate that TASO potentiated T cell-mediated antitumor immunity, and it is proposed that TGF-ß2 may be a promising target in the immunotherapeutic strategy of TNBC.

Artesunate inhibits the development of PVR by suppressing the TGF-ß/Smad signaling pathway.

Proliferative vitreoretinopathy (PVR) is the main cause of retinal detachment surgery failure. The epithelial-mesenchymal transition (EMT) induced by transforming growth factor (TGF-ß2) plays an important role in the development of PVR. Artesunate has been widely studied as a treatment for ophthalmic diseases because of its antioxidant, anti-inflammatory, antiapoptotic and antiproliferative properties. The purpose of this study was to investigate the effects of artesunate on the TGF-ß2-induced EMT in ARPE-19 cells and PVR development. We found that artesunate inhibited the proliferation and contraction of ARPE-19 cells after the EMT and the autocrine effects of TGF-ß2 on ARPE-19 cells. Additionally, the levels of Smad3 and p-Smad3 were increased in clinical samples, and artesunate decreased the levels of Smad3 and p-Smad3 in ARPE-19 cells treated with TGF-ß2. Artesunate also inhibited the occurrence and development of PVR in vivo. In summary, artesunate inhibits the occurrence and development of PVR by inhibiting the EMT in ARPE-19 cells.

Capsaicin attenuates TGFß2-induced epithelial-mesenchymal-transition in lens epithelial cells in vivo and in vitro.

Posterior capsule opacification (PCO), the most common complication of cataract surgery occurring in 20-50% of patients after 2-5 years of cataract surgery, is a major problem in the aging society. The epithelial-mesenchymal transition (EMT) of lens epithelial cells after cataract surgery has been proposed as a major cause of PCO. Capsaicin, widely used as a food additive and analgesic agent, is a major pungent ingredient in red pepper. Although the effect of capsaicin on EMT has been reported in cancer cells, the biological reaction of capsaicin was unique in each cell type, and there have been no reports describing its effects on EMT earlier. In this study, we demonstrated that treatment with capsaicin inhibited TGFß2-induced EMT in vitro lens epithelial cells and ex vivo explant lens epithelial cells. Furthermore, eye drops of capsaicin inhibited the PCO model mice in vivo. Finally, we showed that capsaicin inhibited non-canonically induced Smad2/3 activation via suppression of EGFR activation and ERK phosphorylation. Our findings indicate that capsaicin and its derivatives are good candidate compounds for preventing PCO after cataract surgery.

TGF-ß1 protein trap AVID200 beneficially affects hematopoiesis and bone marrow fibrosis in myelofibrosis.

Myelofibrosis (MF) is a progressive chronic myeloproliferative neoplasm characterized by hyperactivation of JAK/STAT signaling and dysregulation of the transcription factor GATA1 in megakaryocytes (MKs). TGF-ß plays a pivotal role in the pathobiology of MF by promoting BM fibrosis and collagen deposition and by enhancing the dormancy of normal hematopoietic stem cells (HSCs). In this study, we show that MF-MKs elaborated significantly greater levels of TGF-ß1 than TGF-ß2 and TGF-ß3 to a varying degree, and we evaluated the ability of AVID200, a potent TGF-ß1/TGF-ß3 protein trap, to block the excessive TGF-ß signaling. Treatment of human mesenchymal stromal cells with AVID200 significantly reduced their proliferation, decreased phosphorylation of SMAD2, and interfered with the ability of TGF-ß1 to induce collagen expression. Moreover, treatment of MF mononuclear cells with AVID200 led to increased numbers of progenitor cells (PCs) with WT JAK2 rather than mutated JAK2V617F. This effect of AVID200 on MF PCs was attributed to its ability to block TGF-ß1-induced p57Kip2 expression and SMAD2 activation, thereby allowing normal rather than MF PCs to preferentially proliferate and form hematopoietic colonies. To assess the in vivo effects of AVID200, Gata1lo mice, a murine model of MF, were treated with AVID200, resulting in the reduction in BM fibrosis and an increase in BM cellularity. AVID200 treatment also increased the frequency and numbers of murine progenitor cells as well as short-term and long-term HSCs. Collectively, these data provide the rationale for TGF-ß1 blockade, with AVID200 as a therapeutic strategy for patients with MF.

Decorin-An Antagonist of TGF-ß in Astrocytes of the Optic Nerve.

During the pathogenesis of glaucoma, optic nerve (ON) axons become continuously damaged at the optic nerve head (ONH). This often is associated with reactive astrocytes and increased transforming growth factor (TGF-ß) 2 levels. In this study we tested the hypothesis if the presence or absence of decorin (DCN), a small leucine-rich proteoglycan and a natural inhibitor of several members of the TGF family, would affect the expression of the TGF-ßs and connective tissue growth factor (CTGF/CCN2) in human ONH astrocytes and murine ON astrocytes. We found that DCN is present in the mouse ON and is expressed by human ONH and murine ON astrocytes. DCN expression and synthesis was significantly reduced after 24 h treatment with 3 nM CTGF/CCN2, while treatment with 4 pM TGF-ß2 only reduced expression of DCN significantly. Conversely, DCN treatment significantly reduced the expression of TGF-ß1, TGF-ß2 and CTGF/CCN2 vis-a-vis untreated controls. Furthermore, DCN treatment significantly reduced expression of fibronectin (FN) and collagen IV (COL IV). Notably, combined treatment with DCN and triciribine, a small molecule inhibitor of protein kinase B (AKT), attenuated effects of DCN on CTGF/CCN2, TGF-ß1, and TGF-ß2 mRNA expression. We conclude (1) that DCN is an important regulator of TGF-ß and CTGF/CCN2 expression in astrocytes of the ON and ONH, (2) that DCN thereby regulates the expression of extracellular matrix (ECM) components and (3) that DCN executes its negative regulatory effects on TGF-ß and CTGF/CCN2 via the pAKT/AKT signaling pathway in ON astrocytes.

Blockade of transforming growth factor ß2 by anti-sense oligonucleotide improves immunotherapeutic potential of IL-2 against melanoma in a humanized mouse model.

BACKGROUND AIMS: IL-2 is a potent cytokine that activates natural killer cells and CD8+ cytotoxic T lymphocytes (CTLs) and has been approved for the treatment of metastatic renal cell carcinoma and metastatic melanoma. However, the medical use of IL-2 is restricted because of its narrow therapeutic window and potential side effects, including the expansion of regulatory T cells (Tregs). METHODS: In this study, the authors investigated the complementary effects of transforming growth factor-ß2 (TGF-ß2) anti-sense oligodeoxynucleotide (TASO) on the immunotherapeutic potential of IL-2 in a melanoma-bearing humanized mouse model. RESULTS: The authors observed that the combination of TASO and IL-2 facilitated infiltration of CTLs into the tumor, thereby potentiating the tumor killing function of CTLs associated with increased granzyme B expression. In addition, TASO attenuated the increase in Tregs by IL-2 in the peripheral blood and spleen and also inhibited infiltration of Tregs into the tumor, which was partly due to decreased CCL22. Alteration of T-cell constituents at the periphery by TGF-ß2 inhibition combined with IL-2 might be associated with the synergistic augmentation of serum pro-inflammatory cytokines (such as interferon Î³ and tumor necrosis factor α) and decreased ratio of Tregs to CTLs in tumor tissues, which consequently results in significant inhibition of tumor growth CONCLUSIONS: These results indicate that the application of TASO improves IL-2-mediated anti-tumor immunity, thus implying that blockade of TGF-ß2 in combination with IL-2 may be a promising immunotherapeutic strategy for melanoma.

TGF-ß2 interfering oligonucleotides used as adjuvants for microbial vaccines.

The success of using immune checkpoint inhibitors to treat cancers implies that inhibiting an immunosuppressive cytokine, such as TGF-ß2, could be a strategy to develop novel adjuvants for microbial vaccines. To develop nucleic acid based TGF-ß2 inhibitors, we designed three antisense oligonucleotides, designated as TIO1, TIO2, and TIO3, targeting the conserve regions identical in human and mouse TGF-ß2 mRNA 3'-untranslated region. In cultured immune cells, TIO3 and TIO1 significantly reduced the TGF-ß2 mRNA expression and protein production. In mice, the TIO3 and TIO1, when formulated in various microbial vaccines, significantly enhanced the antibody response to the vaccines, and the TIO3-adjuvanted influenza virus vaccine induced effective protection against the influenza virus challenge. In the immunized mice, TIO3 formulated in microbial vaccines dramatically reduced surface-bound TGF-ß2 expression on CD4+ T cells and CD19+ B cells in the lymph node (LN) cells and spleen cells; up-regulated the expression of CD40, CD80, CD86, and MHC II molecules on CD19+ B cells and CD11c+ dendritic cells; and promoted IFN-γ production in CD4+ T cells and CD8+ T cells in the LN cells. Overall, TIO3 or TIO1 could be used as a novel type of adjuvant for facilitating the microbial vaccines to elicit more vigorous and persistent antibody response by interfering with TGF-ß2 expression.

Statins Suppress TGF-ß2-Mediated MMP-2 and MMP-9 Expression and Activation Through RhoA/ROCK Inhibition in Astrocytes of the Human Optic Nerve Head.

Purpose: Matrix metalloproteinases (MMPs) are involved in extracellular matrix (ECM) maintenance and remodeling. The present study aimed to determine whether transforming growth factor (TGF)-ß2 regulates MMP-2 and MMP-9 levels and activities in astrocytes derived from the optic nerve head (ONH) and the role of statins in such modulation. Methods: Primary astrocytes cultured from the lamina cribrosa of human donor ONHs were incubated with three types of statins (5 µg/mL) for 1 hour followed by recombinant TGF-ß2 (5 ng/mL) for various periods to test their effects. Levels and activities of MMP-2 and MMP-9 in astrocytes in vitro were determined by western blotting and zymography, respectively. Levels of phosphorylated myosin phosphatase target subunit 1 (MYPT1) in astrocyte lysates were determined by western blotting, and those of phosphorylated myosin light chain (MLC) were determined by western blotting and immunocytochemistry. Results: MMP-2 and MMP-9 levels were upregulated by TGF-ß2 in human ONH astrocytes. Prior incubation with simvastatin, lovastatin, and atorvastatin inhibited TGF-ß2-mediated MMP-2 and MMP-9 expression and activities. Prior incubation with statins downregulated the TGF-ß2-induced phosphorylation of MYPT1 and MLC, which are downstream substrates of RhoA and ROCKs. Conclusions: Statins inhibited the TGF-ß2-mediated regulation of MMP-2 and MMP-9 by inhibiting the RhoA/ROCK signaling pathway. Considering the role of MMP in ECM remodeling, the present findings support the notion that statins positively impact ECM remodeling within the ONH.

Curcumin Inhibits Proliferation and Epithelial-Mesenchymal Transition in Lens Epithelial Cells through Multiple Pathways.

BACKGROUND: Posterior capsule opacification (PCO), a complication of extracapsular lens extraction surgery that causes visual impairment, is characterized by aberrant proliferation and epithelial-mesenchymal transition (EMT) of lens epithelial cells (LECs). Curcumin, exerting inhibitive effects on cell proliferation and EMT in cancer, serves as a possible antidote towards PCO. METHODS: Cellular proliferation of LECs after treatment of curcumin was measured with MTT assay and flow cytometry. The transcriptional and expressional levels of proteins related to proliferation and EMT of LECs were quantified by western blotting and real-time PCR. RESULTS: Curcumin was found to suppress the proliferation of LECs by inducing G2/M arrest via possible inhibition of cell cycle-related proteins including CDK1, cyclin B1, and CDC25C. It had also inactivated proliferation pathways involving ERK1/2 and Akt pathways in LECs. On the other hand, curcumin downregulated the EMT of LECs through blocking the TGF-ß/Smad pathway and interfering Notch pathway which play important roles in PCO. CONCLUSIONS: This study shows that curcumin could suppress the proliferation and EMT in LECs, and it might be a potential therapeutic protection against visual loss induced by PCO.

Effects of Toll-Like Receptor 4 Inhibition on Transforming Growth Factor-ß2 Signaling in the Human Trabecular Meshwork.

Purpose: Transforming growth factor-ß2 (TGFß2) and Toll-like receptor 4 (TLR4) crosstalk have been implicated in extracellular matrix regulation in the trabecular meshwork (TM) and ocular hypertension in mice. We investigated TLR4 expression in normal and glaucomatous human trabecular meshwork (HTM) sections and utilized a human perfusion organ culture model to determine TGFß2-TLR4 signaling crosstalk in glaucoma. Methods: Expression of TLR4 was determined in TM of normal and glaucomatous human eyes. Anterior segments of paired human eyes were perfused at a constant flow rate (2.5 µL/min) for 4 days to acquire stable baseline intraocular pressures (IOPs). We treated paired eyes with two different treatment paradigms: (1) TGFß2 in one eye and vehicle control in the paired eye, (2) TGFß2 in one eye and TGFß2 + TLR4 inhibitor TAK-242 in the paired eye. Perfusate and TM tissue were collected and analyzed for fibronectin (FN) and collagen IV (COLIV) expression. Results: We observed increased TLR4 expression in glaucomatous HTM sections compared to normal (age-matched) (P < 0.05). Significant elevation of IOP was detected in 47% of TGFß2-treated anterior segments (P < 0.01) compared to control, and in TGFß2 treated compared with co-treatment with TGFß2 + TLR4 inhibitor (P < 0.0001). An increase in FN and COLIV expression was observed after TGFß2 treatment, and inhibition of TLR4 signaling decreased TGFß2-induced FN and COLIV expression in perfusate (P < 0.05). Conclusions: These studies identify TGFß2-TLR4 crosstalk as a novel pathway in glaucoma. They provide a potential new target to lower IOP and explore glaucoma pathogenesis.

Pharmacological Targeting of BET Bromodomains Inhibits Lens Fibrosis via Downregulation of MYC Expression.

Purpose: Lens fibrosis involves aberrant growth, migration, and transforming growth factorß (TGFß)-induced epithelial-mesenchymal transition (EMT) of lens epithelial cells (LECs). In this study, we investigated the role of the bromo- and extra-terminal domain (BET) inhibitor in lens fibrotic disorder to identify drug-based therapies. Methods: Rat lens explants, rabbit primary lens epithelial cells (rLECs), human lens explants and human SRA01/04 cells were treated with TGFß2 in the presence or absence of the BET bromodomain inhibitor JQ1 or the MYC inhibitor 10058-F4. Proliferation was determined by MTS assay. Cell migration was measured by wound healing and transwell assays. The expression levels of fibronectin (FN), α-smooth muscle actin (α-SMA), E-cadherin, and phosphorylated downstream Smads were analyzed by Western blot, qRT-PCR, and immunocytochemical experiments. Transcriptome analysis was conducted to explore the molecular mechanism. Results: Blockage of BET bromodomains with JQ1 significantly suppressed rLECs proliferation by inducing G1 cell cycle arrest. Furthermore, JQ1 attenuated TGFß2-dependent upregulation of mesenchymal gene expression and phosphorylation of Smad2/3 during the progression of EMT, whereas E-cadherin expression was preserved. JQ1 repressed MYC expression, which was dose- and time-dependently upregulated by TGFß2. Inhibiting MYC with either the small-molecule inhibitor 10058-F4 or genetic knockdown phenocopied the effects of JQ1 treatment. MYC overexpression partially reversed the JQ1-regulated EMT-related alteration of gene expression. Both JQ1 and 10058-F4 blocked the expression of TGFß receptor II and integrin αv in rLECs and abolished TGFß2-induced opacification and subcapsular plaque formation in rat lens explants. Conclusions: Our results demonstrate the antifibrotic role of JQ1 in maintaining the epithelial characteristics of LECs and blocking TGFß2-induced EMT, possibly by downregulating MYC, thereby providing new avenues for treating lens fibrosis.

Therapeutic Targeting of TGFß Ligands in Glioblastoma Using Novel Antisense Oligonucleotides Reduces the Growth of Experimental Gliomas.

PURPOSE: Transforming growth factor (TGF)-ß is expressed at high levels by glioma cells and contributes to the malignant phenotype of glioblastoma. However, its therapeutic targeting remains challenging. Here, we examined an alternative therapeutic approach of TGFß inhibition using two novel phosphorothioate-locked nucleic acid (LNA)-modified antisense oligonucleotide gapmers, ISTH1047 and ISTH0047, which specifically target TGFß1 and TGFß2. EXPERIMENTAL DESIGN: We characterized the effects of ISTH1047 and ISTH0047 on TGFß1/2 expression, downstream signaling and growth of human LN-308, LN-229, and ZH-161 cells as well as murine SMA-560 glioma cells in vitro. Furthermore, we assessed their target inhibition and effects on survival in orthotopic xenogeneic and syngeneic rodent glioma models in vivo. RESULTS: Both antisense oligonucleotides specifically silenced their corresponding target and abrogated SMAD2 phosphorylation in several glioma cell lines. Moreover, inhibition of TGFß1 or TGFß2 expression by ISTH1047 or ISTH0047 reduced the migration and invasiveness of LN-308 and SMA-560 glioma cells. Systemic antisense oligonucleotide administration to glioma-bearing mice suppressed TGFß1 or TGFß2 mRNA expression as well as the expression of the downstream target PAI-1 in orthotopic gliomas. Glioma-bearing mice had significantly prolonged survival upon systemic treatment with ISTH1047 or ISTH0047, which was associated with a reduction of intratumoral SMAD2 phosphorylation and, in a fully immunocompetent model, with increased immune cell infiltration. CONCLUSIONS: Targeting TGFß expression with the novel LNA antisense oligonucleotides ISTH1047 or ISTH0047 results in strong antiglioma activity in vitro and in vivo, which may represent a promising approach to be examined in human patients with glioma.

TGF-ß2 antagonizes IL-6-promoted cell survival.

Transforming growth factor beta is a key cytokine involved in the pathogenesis of fibrosis in many organs, whereas interleukin-6 plays an important role in the regulation of inflammation. They are both potent angiogenesis inducers with opposite effects on cell survival and apoptosis. TGF-ß2 induces apoptosis; in contrast, IL-6 protects cells from apoptosis. The possible interaction between these two cytokines is indicated in various disease states. In this study, we have assessed the effect of TGF-ß2 on IL-6 signaling and found that TGF-ß2 could strongly inhibit IL-6-induced STAT3 activation and synergy with IL-6 resulting in enhanced SOCS3 expression. Interestingly, IL-6 also slows down the decay of TGF-ß2 mRNA. Consistent with this mechanism, we found that TGF-ß2 could antagonize IL-6 effect on cell survival in both γ-irradiation and UV light-induced apoptosis. Taken together, the finding shows that TGF-ß2 serves as a negative regulator of IL-6 signaling and antagonizes the anti-apoptosis effect of IL-6.

Effects of Salidroside on Trabecular Meshwork Cell Extracellular Matrix Expression and Mouse Intraocular Pressure.

Purpose: Excessive accumulation of extracellular matrix (ECM) in the trabecular meshwork (TM) reduces aqueous humor outflow, which likely contributes to elevation of IOP in primary open-angle glaucoma (POAG). Salidroside, a phenolic glycoside isolated from Rhodiola rosea is reported to prevent profibrotic responses by inhibiting Smad signaling pathway activated by TGF-ß in liver, lung, and kidney tissues. We tested if salidroside can (1) inhibit TGF-ß2-induced ECM expression in cultured human TM cells, and (2) lower TGF-ß2-induced ocular hypertension in the mouse. Methods: Cultured human TM cells stimulated with 5 ng/mL TGF-ß2 for 48 hours were treated with salidroside for 24 hours. The expressions of fibronectin (FN), collagen type IV (COL-IV), and laminin (LN) were evaluated by quantitative PCR, Western blot, and immunocytochemistry. BALB/cJ mice were injected intravitreally with an adenoviral vector encoding a bioactive mutant of TGF-ß2 (Ad.hTGF-ß2226/228) in one eye to induce ocular hypertension, with the uninjected contralateral or Ad.Empty-injected eyes serving as controls. Mice were treated with a daily intraperitoneal injection of 40 mg/kg salidroside. Conscious mouse IOP values were measured using a TonoLab rebound tonometer. Results: In cultured human TM cells, treatment with TGF-ß2 increased expressions of FN, COL-IV, and LN, as assessed by quantitative PCR, Western blotting, and immunocytochemistry, all of which were significantly and completely ameliorated by 30 µM salidroside. Daily intraperitoneal injections of salidroside (40 mg/kg), starting either at day 0 (same day as Ad.hTGF-ß2226/228 injection) or at day 14, significantly lowered TGF-ß2-induced ocular hypertension in the mouse. In contrast, salidroside did not affect IOP of control eyes. Conclusions: These results demonstrated that salidroside is capable of minimizing TGF-ß2-induced ECM expression in cultured human TM cells. It also reduced TGF-ß2-induced ocular hypertension in the mouse. These findings indicate that this phenolic glycoside may be useful as a novel treatment for POAG.

NEAT1 regulates neuroglial cell mediating Aß clearance via the epigenetic regulation of endocytosis-related genes expression.

The accumulation of intracellular ß-amyloid peptide (Aß) is important pathological characteristic of Alzheimer's disease (AD). However, the exact underlying molecular mechanism remains to be elucidated. Here, we reported that Nuclear Paraspeckle Assembly Transcript 1 (NEAT1), a long n on-coding RNA, exhibits repressed expression in the early stage of AD and its down-regulation declines neuroglial cell mediating Aß clearance via inhibiting expression of endocytosis-related genes. We find that NEAT1 is associated with P300/CBP complex and its inhibition affects H3K27 acetylation (H3K27Ac) and H3K27 crotonylation (H3K27Cro) located nearby to the transcription start site of many genes, including endocytosis-related genes. Interestingly, NEAT1 inhibition down-regulates H3K27Ac but up-regulates H3K27Cro through repression of acetyl-CoA generation. NEAT1 also mediates the binding between STAT3 and H3K27Ac but not H3K27Cro. Therefore, the decrease of H3K27Ac and/or the increase of H3K27Cro declines expression of multiple related genes. Collectively, this study first reveals the different roles of H3K27Ac and H3K27Cro in regulation of gene expression and provides the insight of the epigenetic regulatory mechanism of NEAT1 in gene expression and AD pathology.

Isoform-specific effects of transforming growth factor ß on endothelial-to-mesenchymal transition.

Endothelial-to-mesenchymal transition (EndMT) was first reported in the embryogenesis. Recent studies show that EndMT also occurs in the disease progression of atherosclerosis, cardiac and pulmonary fibrosis, pulmonary hypertension, diabetic nephropathy, and cancer. Although transforming growth factor ß (TGFß) is crucial for EndMT, it is not clear which isoform elicits a predominant effect. The current study aims to directly compare the dose-dependent effects of TGFß1, TGFß2, and TGFß3 on EndMT and characterize the underlying mechanisms. In our results, all three TGFß isoforms induced EndMT in human microvascular endothelial cells after 72 hr, as evidenced by the increased expression of mesenchymal markers N-cadherin and α-smooth muscle actin as well as the decreased expression of endothelial nitric oxide synthase. Interestingly, the effect of TGFß2 was the most pronounced. At 1 ng/ml, only TGFß2 treatment resulted in significantly increased phosphorylation (activation) of Smad2/3 and p38-MAPK and increased expression of mesenchymal transcription factors Snail and FoxC2. Intriguingly, we observed that treatment with 1 ng/ml TGFß1 and TGFß3, but not TGFß2, resulted in an increased expression of TGFß2, thus indicating that EndMT with TGFß1 and TGFß3 treatments was due to the secondary effects through TGFß2 secretion. Furthermore, silencing TGFß2 using small interfering RNA blunted the expression of EndMT markers in TGFß1- and TGFß3-treated cells. Together, our results indicate that TGFß2 is the most potent inducer of EndMT and that TGFß1- and TGFß3-induced EndMT necessitates a paracrine loop involving TGFß2.

Rescue of Premature Coronal Suture Fusion with TGF-ß2 Neutralizing Antibody in Rabbits with Delayed-Onset Synostosis.

OBJECTIVES: An overexpression of Tgf-ß2 leads to calvarial hyperostosis and suture fusion in individuals with craniosynostosis. Inhibition of Tgf-ß2 may help rescue fusing sutures and restore normal growth. The present study was designed to test this hypothesis. DESIGN: Twenty-eight New Zealand White rabbits with delayed-onset coronal synostosis had radiopaque markers placed on either side of the coronal sutures at 10 days of age. The rabbits were randomly assigned to: (1) sham control rabbits (n = 10), (2) rabbits with control IgG (100 µg/suture) delivered in a collagen vehicle (n = 9), and (3) rabbits with Tgf-ß2 neutralizing antibody (100 µg/suture) delivered in a collagen vehicle (n = 9). Longitudinal growth data were collected at 10, 25, 42, and 84 days of age. Sutures were harvested at 84 days of age for histomorphometry. RESULTS: Radiographic analysis showed significantly greater ( P < .05) coronal suture marker separation, craniofacial length, cranial vault length, height, shape indices, cranial base length, and more lordotic cranial base angles in rabbits treated with anti-Tgf-ß2 antibody than in controls at 42 and 84 days of age. Histologically, rabbits treated with anti-Tgf-ß2 antibody at 84 days of age had patent and significantly ( P < .05) wider coronal sutures and greater sutural area compared to controls. CONCLUSIONS: These data support our hypothesis that antagonism of Tgf-ß2 may rescue fusing coronal sutures and facilitate craniofacial growth in this rabbit model. These findings also suggest that cytokine therapy may have clinical significance in infants with progressive postgestational craniosynostosis.

MicroRNA-486-5p suppresses TGF-ß2-induced proliferation, invasion and epithelial-mesenchymal transition of lens epithelial cells by targeting Smad2.

The pathological development of lens epithelial cells (LECs) leads to posterior capsular opacification (PCO). This study was undertaken to investigate the effects of microRNA-486-5p (miR-486-5p) on TGF-ß2-induced proliferation, invasion and epithelial-mesenchymal transition (EMT) in the lens epithelial cell line SRA01/04, and to explore the underlying molecular mechanisms. The expression of miR-486-5p in TGF-ß2-induced SRA01/04 cells was down-regulated, and the expression of Smad2, p-Smad2 and p-Smad3 was up-regulated. A dual-luciferase reporter assay revealed that miR-486-5p directly targets the 30'-UTR of Smad2. MiR-486-5p mimic transfection markedly down-regulated the expression levels of Smad2, thus inhibiting the expression of p-Smad2 and p-Smad3. MiR-486-5p overexpression in SRA01/04 cells markedly suppressed TGF-ß2-induced proliferation and invasion, inhibited protein expression of CDK2 and CDK4, down-regulated fibronectin, α-SMA and vimentin and up-regulated E-cadherin; these effects were partly reversed by Smad2 overexpression. In short, these data show that miR-486-5p overexpression can inhibit TGF-ß2-induced proliferation, invasion and EMT in SRA01/04 cells by repressing Smad2/Smad3 signalling, implying that miR-486-5p may be an effective target to interfere in the progression of PCO.