17ß-estradiol inhibits TGF-ß-induced collagen gel contraction mediated by human Tenon fibroblasts via Smads and MAPK signaling pathways.

AIM: To investigate the impact of 17ß-estradiol on the collagen gels contraction (CGC) and inflammation induced by transforming growth factor (TGF)-ß in human Tenon fibroblasts (HTFs). METHODS: HTFs were three-dimensionally cultivated in type I collagen-generated gels with or without TGF-ß (5 ng/mL), 17ß-estradiol (12.5 to 100 µmol/L), or progesterone (12.5 to 100 µmol/L). Then, the collagen gel diameter was determined to assess the contraction, and the development of stress fibers was analyzed using immunofluorescence staining. Immunoblot and gelatin zymography assays were used to analyze matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) being released into culture supernatants. Enzyme-linked immunosorbent assay (ELISA) and reverse transcription-quantitative polymerase chain reaction (RT-PCR) were used to detect interleukin (IL)-6, monocyte chemoattractant proteins (MCP)-1, and vascular endothelial growth factor (VEGF) in HTFs at the translational and transcriptional levels. The phosphorylation levels of Sma- and Mad-related proteins (Smads), mitogen-activated protein kinases (MAPKs), and protein kinase B (AKT) were measured by immunoblotting. Statistical analysis was performed using either the Tukey-Kramer test or Student's unpaired t-test to compare the various treatments. RESULTS: The CGC caused by TGF-ß in HTFs was significantly inhibited by 17ß-estradiol (25 to 100 µmol/L), and a statistically significant difference was observed when comparing the normal control group with 17ß-estradiol concentrations exceeding 25 µmol/L (P<0.05). The suppressive impact of 17ß-estradiol became evident 24h after administration and peaked at 72h (P<0.05), whereas progesterone had no impact. Moreover, 17ß-estradiol attenuated the formation of stress fibers, and the production of MMP-3 and MMP-1 in HTFs stimulated by TGF-ß. The expression of MCP-1, IL-6, and VEGF mRNA and protein in HTFs were suppressed by 100 µmol/L 17ß-estradiol (P<0.01). Additionally, the phosphorylation of Smad2 Smad3, p38, and extracellular signal-regulated kinase (ERK) were downregulated (P <0.01). CONCLUSION: 17ß-estradiol significantly inhibits the CGC and inflammation caused by TGF-ß in HTFs. This inhibition is likely related to the suppression of stress fibers, inhibition of MMPs, and attenuation of Smads and MAPK (ERK and p38) signaling. 17ß-estradiol may have potential clinical benefits in preventing scar development and inflammation in the conjunctiva.

Protection of Barrier Function in Cultured Human Corneal Epithelial Cells by Semaphorin 4D.

PURPOSE: Corneal epithelial barrier function is important to maintain corneal homeostasis and is impaired by inflammation. We aimed to investigate the localization of semaphorin 4D (Sema4D) in the cornea, and its effects on the barrier function of cultured corneal epithelial cells. METHODS: The expressions of semaphorin4 D and its receptor in the murine cornea were examined by immunoblot, immunofluorescent staining and confocal microscopy observations. Human corneal epithelial (HCE) cells stimulated by TNF-α or IL-1ß were cultured with or without Sema4D. Cell viability was examined by CCK8, cell migration was evaluated by scratch wound assay, and barrier function was assessed by transepithelial electrical resistance (TEER) and Dextran-FITC permeability assay. The expression of tight junction proteins in HCE cells was examined by immunoblot, immunofluorescent staining and qRT-PCR. RESULTS: We demonstrated that the protein of Sema4D and its receptor, plexin-B1, was expressed in murine cornea. Sema4D induced an increase in the TEER and a decrease in the permeability of HCE cells. It also induced the expression of tight junction protein ZO-1, occludin and claudin-1 in HCE cells. Furthermore, under stimulation of TNF-α or IL-1ß, Sema4D treatment could inhibit the decreased TEER and the elevated permeability of HCE cells. CONCLUSIONS: Sema4D is located distinctly in corneal epithelial cells and promoted their barrier function by increasing the expression of tight junction proteins. Sema4D may act as a preventive for maintaining corneal epithelial barrier function during ocular inflammation.

Luteolin ameliorates cornea stromal collagen degradation and inflammatory damage in rats with corneal alkali burn.

Corneal alkali burn (AB) is a blindness-causing ocular trauma commonly seen in clinics. An excessive inflammatory reaction and stromal collagen degradation contribute to corneal pathological damage. Luteolin (LUT) has been studied for its anti-inflammatory effects. In this study, the effect of LUT on cornea stromal collagen degradation and inflammatory damage in rats with corneal alkali burn was investigated. After corneal alkali burn, rats were randomly assigned to the AB group and AB + LUT group and received an injection of saline and LUT (200 mg/kg) once daily. Subsequently, corneal opacity, epithelial defects, inflammation and neovascularization (NV) were observed and recorded on Days 1, 2, 3, 7 and 14 post-injury. The concentration of LUT in ocular surface tissues and anterior chamber, as well as the levels of collagen degradation, inflammatory cytokines, matrix metalloproteinases (MMPs) and their activity in the cornea were detected. Human corneal fibroblasts (HCFs) were co-cultured with interleukin (IL)-1ß and LUT. Cell proliferation and apoptosis were assessed by CCK-8 assay and flow cytometry respectively. Measurement of hydroxyproline (HYP) in culture supernatants was used to quantify the amount of collagen degradation. Plasmin activity was also assessed. ELISA or real-time PCR was used to detect the production of matrix metalloproteinases (MMPs), IL-8, IL-6 and monocyte chemotactic protein (MCP)-1. Furthermore, the immunoblot method was used to assess the phosphorylation of mitogen-activated protein kinases (MAPKs), transforming growth factor-ß-activated kinase (TAK)-1, activator protein-1 (AP-1) and inhibitory protein IκB-α. At last, immunofluorescence staining helped to develop nuclear factor (NF)-κB. LUT was detectable in ocular tissues and anterior chamber after intraperitoneal injection. An intraperitoneal injection of LUT ameliorated alkali burn-elicited corneal opacity, corneal epithelial defects, collagen degradation, NV, and the infiltration of inflammatory cells. The mRNA expressions of IL-1ß, IL-6, MCP-1, vascular endothelial growth factor (VEGF)-A, and MMPs in corneal tissue were downregulated by LUT intervention. And its administration reduced the protein levels of IL-1ß, collagenases, and MMP activity. Furthermore, in vitro study showed that LUT inhibited IL-1ß-induced type I collagen degradation and the release of inflammatory cytokines and chemokines by corneal stromal fibroblasts. LUT also inhibited the IL-1ß-induced activation of TAK-1, mitogen-activated protein kinase (MAPK), c-Jun, and NF-κB signaling pathways in these cells. Our results demonstrate that LUT inhibited alkali burn-stimulated collagen breakdown and corneal inflammation, most likely by attenuating the IL-1ß signaling pathway. LUT may therefore prove to be of clinical value for treating corneal alkali burns.

Sulforaphane inhibits TGF-ß-induced fibrogenesis and inflammation in human Tenon's fibroblasts.

Purpose: Subconjunctival fibrosis is the main cause of failure after glaucoma filtration surgery. We explored the effects of sulforaphane (SFN) on the conversion of human Tenon's fibroblasts (HTFs) into myofibroblasts, transforming growth factor (TGF)-ß-induced contraction of collagen gel, and inflammation. Methods: After treatment with the combination of TGF-ß and SFN or TGF-ß alone, primary HTFs were subjected to a three-dimensional collagen contraction experiment to examine their contractility. Levels of α smooth muscle actin (α-SMA), synthesis of extracellular matrix (ECM), and phosphorylation of various signaling molecules were determined by western blot or quantitative reverse transcription-polymerase chain reaction (RT-qPCR). Fluorescence microscopy was employed to examine stress fiber formation in HTFs. The expressions of interleukin (IL)-6, IL-8, and connective tissue growth factor (CTGF) were determined using RT-qPCR. Results: The contraction of myofibroblasts caused by TGF-ß was significantly suppressed by SFN. This suppressive effect was exerted via the differentiation of HTFs into myofibroblasts by inhibiting the production of fibronectin and the expression of α-SMA. Moreover, SFN treatment reduced the expression of TGF-ß-promoted integrins ß1 and α5, myosin light chain (MLC) phosphorylation, and stress fiber formation, as well as the expression of IL-6, IL-8, and CTGF. Finally, TGF-ß-induced Smad2/3 and extracellular signal-regulated kinase (ERK) phosphorylations were attenuated by SFN. Conclusions: SFN inhibits HTF contractility, differentiation into myofibroblasts, and inflammation caused by TGF-ß. These effects are mediated by both classic and non-classic signaling pathways. Our results indicate that SFN has potent anti-fibrotic and anti-inflammatory effects in HTFs and is a potential candidate for subconjunctival fibrosis therapy.

Numb deficiency impairs retinal structure and visual function in mice.

PURPOSE: Numb is expressed in the progenitor and mature neurons throughout the development progress of the retina. We attempted to investigate the role of Numb in the retina and visual system, and the effects of Numb deficiency on retinal structure and visual function. METHODS: Conditional Numb/Nbl double-knockout mice were generated to observe the retinal damage in Numb deficiency mice. HE staining and immunofluorescence stain were used to observe the structural and molecular changes. The visual function was assessed by electroretinogram (ERG) and optomotor response (OMR). RNA-Seq and RT-PCR were used to detect the differential expression of genes and the related signaling pathways. RESULTS: Inactivation of Numb/Nbl induces eye apoptosis and retinal neurons impairment observed by HE staining and immunofluorescence stain. The impaired retinal structure and visual function were assessed by ERG and OMR. RNA-seq analysis indicated loss of photoreceptors, synapses and phototransduction related molecules. Immunofluorescence stain of molecular markers recoverin, arrestin, rhodopsin showed disrupted structural integrity of photoreceptors. Additional bipolar cells and synapses related molecular markers indicate synaptic connections were damaged in Numb deficiency mice. CONCLUSIONS: Inactivation of Numb/Nbl induces eye apoptosis and retinal neurons impairment. Ablation of Numb in retina significantly impaired visual function. The impaired visual function in Numb deficiency mice is related to the damage of photoreceptors, ion/cation channel activity, synapse formation and phototransduction.

Sox2 knockdown in the neonatal retina causes cell fate to switch from amacrine to bipolar.

Transcription factor Sox2 is widely recognized for its critical roles in the nervous system, including the neural retina. Here, we aimed to reveal the function of Sox2 in the process of mouse postnatal development. After the suppression of Sox2 at P0, there was an increase number in bipolar cells but a decrease in amacrine cells. Inhibited Sox2 expression also led to decreased visual function. Furthermore, we found a distinctive type of retinal cells expressing the characteristic proteins of both bipolar cells and amacrine cells at P6, which may be an intermediate state in which amacrine cells were transforming into bipolar cells. Transcription factors associated with the development of bipolar cells and amacrine cells also support those changes. Our work indicated that inhibition of Sox2 could change cell fate by affecting transcription factors in the development of bipolar cells and amacrine cells, may provide new directions for the study and treatment of retinal genetic diseases and retinal dysplasia.

Course-, dose-, and stage-dependent toxic effects of prenatal dexamethasone exposure on long bone development in fetal mice.

Dexamethasone is routinely used for treating those mothers at risk for preterm delivery. However, overexposure to exogenous glucocorticoids induces bone loss in offspring, and the "critical window" and safe dose of this treatment are largely unknown. In this study, we found that femoral length, and the length of the primary ossification center were significantly reduced in fetal mice after repeated prenatal dexamethasone exposure (PDE). Compared with single-course exposure on gestational day (GD)15, newborn mice with repeated PDE (3 times, from GD15 to 17) showed a significant decrease in femoral trabecular bone mass with decreased trabecular number and thickness. For those newborn mice treated after repeated PDE at different doses (0, 0.2, 0.8, and 1.2 mg/kg/d), the toxic effect of dexamethasone on bone development was observed at 0.8 and 1.2 mg/kg/d. More severe retardation in bone development was observed in the fetal mice after PDE at 0.8 mg/kg/d during GD12-14, compared with that during GD15-17. Interestingly, stronger toxic effects were observed in male newborn mice after PDE than were observed in female newborn mice. In conclusion, PDE with multiple course, higher dose, or exposure at an early stage of pregnancy have stronger toxic effects on bone development of fetal mice.

Course-, dose-, and stage-dependent toxic effects of prenatal dexamethasone exposure on fetal articular cartilage development.

Dexamethasone, a synthetic long-acting glucocorticoid, is routinely used for treating mothers at risk for preterm delivery. However, intrauterine overexposure to glucocorticoids induces low birth weight and cartilage dysplasia in offspring. Also, the "critical window" and safe dose of this treatment are largely unknown. This study investigated the course-, dose-, and stage-dependent toxic effects and the possible mechanisms of prenatal dexamethasone exposure (PDE) on fetal development and articular cartilage development. Pregnant mice (C57BL/6) received subcutaneous injection of dexamethasone (0.8 mg/kg d) once on gestational day (GD) 15 or once a day from GD 15 to 17, or received various doses of dexamethasone (0, 0.2, 0.8, and 1.2 mg/kg d) on GD 15-17, or received dexamethasone (0.8 mg/kg d) at early stage (GD 12-14) or late stage of pregnancy (GD 15-17). Offspring's knee joints were harvested at birth for morphological analyses and detection of gene expression. Repeated PDE significantly suppressed fetal and articular cartilage development, which were characterized by decreased body weight and body length, coarse articular cartilage surfaces, and reduced gene and protein expression of Col2a1 and aggrecan. For those newborns treated with repeated PDE at different doses, the toxic effects on fetal and articular cartilage development were observed at doses of 0.8 and 1.2 mg/kg d, whereas no obvious toxic effects were observed at the dose of 0.2 mg/kg d. Moreover, PDE at 0.8 mg/kg d during the early embryonic stage induced stronger toxic effects on fetal and articular cartilage development, compared with PDE during the late embryonic stage. Detection of gene expression showed that the TGFß signaling pathway in the articular cartilage was down-regulated after PDE. Taken together, PDE induces fetal developmental toxicity and articular cartilage developmental toxicity in a course-, dose-, and stage-dependent manner.