Induction of meibocyte differentiation by three-dimensional, matrigel culture of immortalized human meibomian gland epithelial cells to form acinar organoids.

PURPOSE: Recent studies have shown that two-dimensional (2D) culture of primary rabbit and immortalized human meibomian gland epithelial cells (iHMGEC) do not recapitulate normal meibocyte differentiation and fail to express critical enzymes necessary for synthesis of meibum lipids. The purpose of this study was to test the hypothesis that 3D-spheroid culture of iHMGEC can facilitate meibocyte differentiation and induce the expression of acyl-CoA wax-alcohol acyltransferase 2 (AWAT2), shown to be required for synthesis of meibum wax esters. METHODS: iHMGEC were suspended in matrigel/basement membrane matrix and grown in proliferation media to form distinct cell clusters or spheroids. Cells were then treated with serum-free, differentiation media (advanced DMEM/F12) with and without FGF10 and synthetic agonists for the nuclear lipid receptor, peroxisome proliferator activator receptor gamma (PPARγ). Cells were then evaluated for differentiation markers using western blotting, immunocytochemistry (ICC) and real-time PCR. Control cells were grown in standard 2D culture systems. RESULTS: Under proliferative conditions, 3D culture induced the formation of KRT5+ spheroids that contained a Ki67+/P63+ undifferentiated, basal cell population. When spheroids were switched to differentiation media containing PPARγ agonists, two different organoid populations were detected, a KRT6low population that was AWAT2+/PPARγ+ and a KRT6high population that was AWAT2-/PPARγ-, suggesting that iHMGEC exhibit a dual differentiation potential toward either a ductal or meibocyte organoid phenotype. CONCLUSION: The 3D culturing of iHMGEC can induce the formation of both meibocyte and ductal organoids and may thus serve as a better in vitro model system for studying the regulatory mechanisms controlling meibomian gland function.

Lobeglitazone attenuates fibrosis in corneal fibroblasts by interrupting TGF-beta-mediated Smad signaling.

PURPOSE: Transforming growth factor beta 1 (TGF-ß1) is an important cytokine released after ocular surface injury to promote wound healing. However, its persistence at the injury site triggers a fibrotic response that leads to corneal scarring and opacity. Thiazolidinediones (TZDs) are synthetic peroxisome proliferator-activated receptor gamma (PPAR-γ) ligands used to regulate glucose and lipid metabolism in the management of type 2 diabetes. Studies have also showed TZDs have antifibrotic effect. In this study, we investigated the antifibrotic effect of the TZD lobeglitazone on TGF-ß1-induced fibrosis in corneal fibroblasts. METHODS: Human primary corneal fibroblasts were cultivated and treated with TGF-ß1 (5 ng/mL) to induce fibrosis, with or without pre-treatments with different concentrations of lobeglitazone. Myofibroblast differentiation and extracellular matrix (ECM) protein expression was evaluated by western blotting, immunofluorescence, real-time PCR, and collagen gel contraction assay. The effect of lobeglitazone on TGF-ß1-induced reactive oxygen species (ROS) generation was evaluated by DCFDA-cellular ROS detection assay kit. Signaling proteins were evaluated by western blotting to determine the mechanism underlying the antifibrotic effect. RESULTS: Our results showed lobeglitazone attenuated TGF-ß1-induced ECM synthesis and myofibroblast differentiation of corneal fibroblasts. This antifibrotic effect appeared to be independent of PPAR signaling and rather due to the inhibition of the TGF-ß1-induced Smad signaling. Lobeglitazone also blocked TGF-ß1-induced ROS generation and nicotinamide adenine dinucleotide phosphate oxidase (Nox) 4 transcription. CONCLUSION: These findings indicate that lobeglitazone may be a promising therapeutic agent for corneal scarring. KEY MESSAGES.

AMPK Activation by 5-Amino-4-Imidazole Carboxamide Riboside-1-ß-D-Ribofuranoside Attenuates Alkali Injury-Induced Corneal Fibrosis.

Purpose: Increased TGF-ß1 synthesis after corneal alkali injury is implicated in corneal fibrosis, as it promotes transdifferentiation of keratocytes into myofibroblasts. The activation of 5'-adenosine monophosphate-activated protein kinase (AMPK) by 5-amino-4-imidazole carboxamide riboside-1-ß-d-ribofuranoside (AICAR) inhibits TGF-ß1-induced fibrosis in other cell types. We investigated the antifibrotic effect of AICAR in corneal fibroblasts after alkali injury. Methods: Mouse models of corneal alkali burn, produced by placing 2-mm-diameter filter paper soaked in 0.1-N NaOH on the right cornea for 30 seconds, were treated with the test drugs 4× daily for 21 days. The central cornea was scanned by optical coherence tomography (OCT). Corneal tissues were obtained and processed for western blotting and immunohistochemistry. For in vitro analysis, primary human corneal fibroblasts were treated directly with TGF-ß1 to induce fibrosis, with or without AICAR pretreatment. Myofibroblast activation and extracellular matrix (ECM) protein synthesis were detected by western blotting, real-time PCR, and collagen gel contraction assay. Signaling proteins were analyzed by western blotting. Results: Alkali injury induced the upregulation of TGF-ß1 expression, which led to increased α-smooth muscle actin (α-SMA) and fibronectin synthesis and myofibroblast differentiation. AMPK activation by AICAR significantly suppressed TGF-ß1 and ECM protein expression. The antifibrotic effect of AICAR was AMPK dependent, as treatment with the AMPK inhibitor Compound C attenuated the antifibrotic response. Conclusions: AMPK activation by AICAR suppresses the myofibroblast differentiation and ECM synthesis that occur after alkali injury in corneal fibroblasts.

Critical Role of mTORC2-Akt Signaling in TGF-ß1-Induced Myofibroblast Differentiation of Human Pterygium Fibroblasts.

Purpose: Profibrotic activation is essential for pterygium development. In this study, we investigated the role of the mechanistic target of rapamycin (mTOR) in regulating TGF-ß1-induced myofibroblastic responses in human pterygium fibroblasts (HPFs) and elucidated the relative contributions of mTOR signaling components. Methods: HPFs were pretreated with the mTOR inhibitors rapamycin and Torin2, and TGF-ß1-induced expression of profibrotic markers, including α-smooth muscle actin (α-SMA) and fibronectin, was evaluated. RNA interference-based approaches targeting raptor and rictor, regulatory subunits of mTOR complex 1 (mTORC1) and 2 (mTORC2), respectively, were used to determine the impact of each mTOR complex on HPFs. The contractile phenotype of HPFs was assessed by a collagen gel contraction assay. Results: The mTOR active-site inhibitor Torin2, which suppresses both mTORC1 and mTORC2 activity in HPFs, inhibited TGF-ß1-induced expression of α-SMA and fibronectin. The allosteric inhibitor rapamycin only partially suppressed mTORC1 activity and exhibited a minimal effect on the induction of profibrotic markers. The induction of α-SMA and fibronectin in HPFs was abrogated by RNA interference-mediated knockdown of rictor but was only moderately affected by raptor knockdown. Akt inhibition mimicked the effect of Torin2 and rictor knockdown on myofibroblast differentiation of HPFs. mTOR inhibition potently reduced the contractile ability of HPFs in collagen gel contraction assays. Conclusions: This study found that mTOR signaling promoted profibrotic activation of HPFs and confirmed the importance of the mTORC2-Akt axis in TGF-ß1-induced myofibroblast differentiation. Therefore, our study may open up new avenues for the development of novel therapeutic strategies involving targeting of mTOR signaling to treat pterygium.

A PPAR-Gamma Agonist Rosiglitazone Suppresses Fibrotic Response in Human Pterygium Fibroblasts by Modulating the p38 MAPK Pathway.

Purpose: Fibroblast activation may play an important role in pterygium progression. Synthetic peroxisome proliferator-activated receptor γ (PPAR-γ) ligands have been shown to be effective antifibrotic agents against transforming growth factor ß1 (TGF-ß1) induced fibrosis in several tissues. We aimed to investigate the antifibrotic effects of the PPAR-γ ligand rosiglitazone in pterygium fibroblasts and the underlying mechanisms. Methods: Profibrotic activation was induced by TGF-ß1 in primary cultured human pterygium fibroblasts and the effect of rosiglitazone treatment on α-smooth muscle actin (α-SMA), and extra cellular matrix proteins synthesis was detected by western blotting, real-time PCR, immunostaining, and flow cytometry. Pharmaceutical inhibition of PPAR-γ receptor was used to determine the dependency or otherwise of rosiglitazone's action on PPAR-γ signaling. Major signaling pathways downstream of TGF-ß1 were investigated by western blotting to assess their possible association with rosiglitazone's effect. Cell viability and apoptosis were investigated to assess drug-induced cytotoxicity, and the effect of rosiglitazone treatment on cell migration was further determined. Results: α-SMA and fibronectin synthesis induced by TGF-ß1 were suppressed by rosiglitazone treatment in a dose-dependent manner. Rosiglitazone also inhibited intrinsic TGF-ß1 expression. Smad2/3, ERK1/2, and P38 pathways were activated in response to TGF-ß1. Rosiglitazone suppressed TGF-ß1-induced P38 MAPK activation, while ERK1/2 and Smad2/3 signaling remained unaffected. The observed antifibrotic effect of rosiglitazone was not affected by the PPAR-γ antagonist GW9662, indicating it is not PPAR-γ dependent. Rosiglitazone also inhibited the proliferation and migration of pterygium fibroblasts. Conclusions: Rosiglitazone suppresses TGF-ß1-induced myofibroblast activation and extra cellular matrix synthesis in pterygium fibroblasts at least partly through the modulation of the p38 MAPK pathway.

Potential therapeutic effect of alkaline reduced water in polycystic ovarian syndrome.

Polycystic ovarian syndrome (PCOS) is an endocrine-metabolic disorder characterized by hormonal disturbances including hyperandrogenemia, insulin resistance, and hyperinsulinemia culminating into obesity, multiple ovarian cysts and anovulatory infertility in women. There has been no effective medication against PCOS and its complication. However, weight loss can reduce insulin resistance, which in turn helps to restore hormonal balance and ovulation resulting to improved fertility. Previously, we reported that alkaline reduced water (ARW) could significantly reduce obesity by alleviating adiposity, regulating the levels of adipokines/pro-inflammatory cytokines and by inducing cholesterol homeostasis. Herein, we hypothesize that ARW might ameliorate the pathophysiological (hormonal, metabolic, and immunological) imbalances incurred by PCOS, thereby improving the infertility of PCOS patients.