Autophagy is induced by raptor degradation via the ubiquitin/proteasome system in granular corneal dystrophy type 2.

Granular corneal dystrophy type 2 (GCD2) is an autosomal dominant disorder that is caused by a point mutation in transforming growth factor-ß-induced gene-h3 (TGFBI), which encodes transforming growth factor-ß-induced protein (TGFBIp). Recently, we found that the autophagic clearance of mutant-TGFBIp is delayed in GCD2 corneal fibroblasts; however, any potential correlation between mutant-TGFBIp turnover and autophagy-lysosome pathway remains unknown. Here, we report that mutant-TGFBIp is accumulated and that autophagy, a key clearance pathway for mutant-TGFBIp, is induced in primary cultured GCD2 homozygous (HO) and wild-type (WT) corneal fibroblasts that express exogenously introduced mutant-TGFBIp. Mutant-TGFBI colocalized with LC3-enriched cytosolic vesicles and cathepsin D in primary cultured GCD2 corneal fibroblasts. We also observed reduced levels of raptor (regulatory-associated protein of the mammalian target of rapamycin [mTOR]) in GCD2 corneal fibroblasts and WT corneal fibroblasts expressing mutant-TGFBIp. Strikingly, treatment with MG132, a ubiquitin/proteasome system inhibitor, significantly increased the levels of both total and ubiquitinated raptor in GCD2 corneal fibroblasts. The levels of the autophagy marker LC3-II were also increased in WT corneal fibroblasts that were treated with shRNA against raptor. However, mutant-TGFBIp accumulated in autophagosomes or/and lysosomes in spite of the significant activation of basal autophagy in GCD2 corneal fibroblasts. These results suggest that an insufficient autophagy-lysosome pathway might be responsible for the intracellular accumulation of mutant-TGFBIp during the pathogenesis of GCD2.

Melatonin induces autophagy via an mTOR-dependent pathway and enhances clearance of mutant-TGFBIp.

The hallmark of granular corneal dystrophy type 2 (GCD2) is the deposit of mutant transforming growth factor-ß (TGF-ß)-induced protein (TGFBIp) in the cornea. We have recently shown that there is a delay in autophagic degradation of mutant-TGFBIp via impaired autophagic flux in GCD2 corneal fibroblasts. We hypothesized that melatonin can specifically induce autophagy and consequently eliminate mutant-TGFBIp in GCD corneal fibroblasts. Our results show that melatonin activates autophagy in both wild-type (WT) and GCD2-homozygous (HO) corneal fibroblast cell lines via the mammalian target of rapamycin (mTOR)-dependent pathway. Melatonin treatment also led to increased levels of beclin 1, which is involved in autophagosome formation and maturation. Furthermore, melatonin significantly reduced the amounts of mutant- and WT-TGFBIp. Treatment with melatonin counteracted the autophagy-inhibitory effects of bafilomycin A1, a potent inhibitor of autophagic flux, demonstrating that melatonin enhances activation of autophagy and increases degradation of TGFBIp. Cotreatment with melatonin and rapamycin, an autophagy inducer, had an additive effect on mutant-TGFBIp clearance compared to treatment with either drug alone. Treatment with the selective melatonin receptor antagonist luzindole did not block melatonin-induced autophagy. Given its ability to activate autophagy, melatonin is a potential therapeutic agent for GCD2.

Decreased catalase expression and increased susceptibility to oxidative stress in primary cultured corneal fibroblasts from patients with granular corneal dystrophy type II.

Granular corneal dystrophy type II (GCD II) is an autosomal dominant disorder characterized by age-dependent progressive accumulation of transforming growth factor-beta-induced protein (TGFBIp) deposits in the corneal stroma. Several studies have suggested that corneal fibroblasts may decline with age in response to oxidative stress. To investigate whether oxidative stress is involved in the pathogenesis of GCD II, we assayed antioxidant enzymes, oxidative damage, and susceptibility to reactive oxygen species-induced cell death in primary cultured corneal fibroblasts (PCFs) from GCD II patients and healthy subjects. We found elevated protein levels of Mn-superoxide dismutase, Cu/Zn-superoxide dismutase, glutathione peroxidase, and glutathione reductase, as well as increased CAT mRNA and decreased catalase protein in GCD II PCFs. Furthermore, catalase is down-regulated in normal PCFs transfected with transforming growth factor-beta-induced gene-h3. We also observed an increase in not only intracellular reactive oxygen species and H(2)O(2) levels, but also malondialdehyde, 4-hydroxynonenal, and protein carbonyls levels in GCD II PCFs. Greater immunoreactivity for malondialdehyde was observed in the corneal tissue of GCD II patients. In addition, we observed a decrease in Bcl-2 and Bcl-xL levels and an increase in Bax and Bok levels in GCD II PCFs. Finally, GCD II PCFs are more susceptible to H(2)O(2)-induced cell death. Together, these results suggest that oxidative damage induced by decreased catalase is involved in GCD II pathogenesis, and antioxidant agents represent a possible treatment strategy.

Evaluation of programmed cell death protein 1 (PD-1) expression as a prognostic biomarker in patients with clear cell renal cell carcinoma.

Programmed cell death protein 1 (PD-1) immune checkpoint inhibitors have shown activity in patients with advanced renal cell carcinoma (RCC). However, the role of PD-1 expression in tumor-infiltrating lymphocytes (TILs) as a biomarker for poor outcome is not clear. In this study, we evaluated the prognostic value of TIL PD-1 expression in patients with clear cell RCC (ccRCC). 82 patients who underwent nephrectomy for localized or metastatic ccRCC and followed up for at least four years were searched from our database and retrospectively enrolled. Their fixed primary tumor specimens were stained with anti-PD-1 (NAT105). The specimens were classified as negative or positive for PD-1 expression, and the positive specimens were further scored in 10% increments. 37 (45.12%) patients were negative (<1% stained), 26 (31.71%) patients were low (<10 and 10%), and 19 (23.17%) patients were high (20-50%) for PD-1 expression. The prognostic value of TIL PD-1 expression was evaluated by univariate Cox proportional hazards regression on overall and recurrence-free survivals. Higher TIL PD-1 expression was not associated with increased risk of death (P = 0.336) or with increased risk of recurrence (P = 0.572). Higher primary tumor stage was associated with increased risk of recurrence (P = 0.003), and higher Fuhrman nuclear grade was associated with increased risk of death (P <0.001) and with increased risk of recurrence (P <0.001). Our study shows that TIL PD-1 expression by immunohistochemistry (IHC) does not correlate with poor clinical outcome in patients with ccRCC and is inferior to established prognosticating tools.

Histone methylation levels correlate with TGFBIp and extracellular matrix gene expression in normal and granular corneal dystrophy type 2 corneal fibroblasts.

BACKGROUND: TGFß1-induced expression of transforming growth factor ß-induced protein (TGFBIp) and extracellular matrix (ECM) genes plays a major role in the development of granular corneal dystrophy type 2 (GCD2: also called Avellino corneal dystrophy). Although some key transcription factors are known, the epigenetic mechanisms modulating TGFBIp and ECM expression remain unclear. We examined the role of chromatin markers such as histone H3 lysine methylation (H3Kme) in TGFß1-induced TGFBIp and ECM gene expression in normal and GCD2-derived human corneal fibroblasts. METHODS: Wild-type (n = 3), GCD2-heterozygous (n = 1), and GCD2-homozygous (n = 3) primary human corneal fibroblasts were harvested from human donors and patients prepared. Microarray and gene-expression profiling, Chromatin immunoprecipitation microarray analysis, and Methylated DNA isolation assay-assisted CpG microarrays was performed in Wild-type and GCD2-homozygous human cells. RESULTS: Transcription and extracellular-secretion levels of TGFBIp were high in normal cells compared with those in GCD2-derived cells and were related to H3K4me3 levels but not to DNA methylation over the TGFBI locus. TGFß1 increased the expression of TGFBIp and the ECM-associated genes connective tissue growth factor, collagen-α2[Ι], and plasminogen activator inhibitor-1 in normal corneal fibroblasts. Increased levels of gene-activating markers (H3K4me1/3) and decreased levels of repressive markers (H3K27me3) at the promoters of those gene accompanied the changes in expression. TGFß1 also increased recruitment of the H3K4 methyltransferase MLL1 and of SET7/9 and also the binding of Smad3 to the promoters. Knockdown of both MLL1 and SET7/9 significantly blocked the TGFß1-induced gene expression and inhibited TGFß1-induced changes in promoter H3K4me1/3 levels. Those effects were very weak, however, in GCD2-derived corneal fibroblasts. CONCLUSIONS: Taken together, the results show the functional role of H3K4me in TGFß1-mediated TGFBIp and ECM gene expression in corneal fibroblasts. Pharmacologic and other therapies that regulate these modifications could have potential cornea-protective effects for granular corneal dystrophy.

Inhibitory Effect of Tranilast on Transforming Growth Factor-Beta-Induced Protein in Granular Corneal Dystrophy Type 2 Corneal Fibroblasts.

PURPOSE: To investigate the effects of tranilast, an inhibitor of chemical mediators and fibroblast proliferation, on the expression of transforming growth factor-beta (TGF-ß)-induced protein (TGFBIp) in wild-type (WT) and homozygous (HO) granular corneal dystrophy type 2 corneal fibroblasts. METHODS: Cell proliferation and cytotoxicity were measured by Cell Counting Kit-8 and lactate dehydrogenase assay. Western blotting and real-time polymerase chain reaction were used to determine changes in the expression of TGFBIp and TGFBI mRNA. We determined the effects of tranilast on phosphorylated Smad2 (pSmad2) and pSmad3, wound-healing, and expression of alpha-smooth muscle actin (α-SMA), type I collagen, and integrins. RESULTS: High concentrations of tranilast decreased proliferation of corneal fibroblasts but did not cause elevation of lactate dehydrogenase, except at 1.0 mM tranilast. TGF-ß increased the expression of TGFBIp and TGFBI mRNA in WT and HO corneal fibroblasts. Cotreatment of corneal fibroblasts with tranilast and TGF-ß reduced the levels of TGFBIp and TGFBI mRNA. In addition, application of tranilast reduced pSmad2 in WT and HO corneal fibroblasts and pSmad3 in HO corneal fibroblasts, both of which were increased initially by TGF-ß. Tranilast delayed wound healing and reduced the expression of α-SMA, type I collagen, and some of integrins in WT and HO corneal fibroblasts. CONCLUSIONS: Application of tranilast in WT and HO corneal fibroblasts inhibited the expression of TGFBIp by blocking TGF-ß signaling. Thus, tranilast may be useful in delaying or preventing the recurrence of corneal opacity in TGFBI-linked corneal dystrophies if clinical studies confirm these findings.

Effects of topical loteprednol etabonate on tear cytokines and clinical outcomes in moderate and severe meibomian gland dysfunction: randomized clinical trial.

PURPOSE: To assess tear cytokine levels and clinical outcomes in moderate and severe meibomian gland dysfunction (MGD) after 2 months of treatment with topical loteprednol etabonate and eyelid scrubs with warm compresses vs eyelid scrubs with warm compresses alone. DESIGN: Randomized controlled trial. METHODS: Patients with moderate and severe MGD were randomized into 2 groups: topical loteprednol etabonate and eyelid scrubs with warm compresses (Group I, 34 eyes) or eyelid scrubs with warm compresses (Group II, 36 eyes). We evaluated cytokine levels, tear film break-up time (TBUT), corneal and conjunctival fluorescein staining, biomicroscopic examination of lid margins and meibomian glands, and the Ocular Surface Disease Index before initiating treatment and 1 month and 2 months after treatment. RESULTS: There were significant decreases in the levels of interleukin (IL)-6, IL-8, and IL-1ß in Group I, and IL-6 and IL-8 in Group II. Moreover, the observed decreases of these cytokines in Group I were attributed to a remarkable decrease between treatment and 1 month after treatment. In Group I, there were improvements in all of the clinical outcomes, with prominent improvement in TBUT, corneal and conjunctival fluorescein staining, and meibum quality after 1 month of treatment, compared with Group II. An improvement in meibomian gland expressibility and MGD stage reduction were more remarkable in Group I. CONCLUSIONS: Topical loteprednol etabonate and eyelid scrubs with warm compresses were tolerated and efficacious for the treatment of moderate and severe MGD. We suggest that such beneficial effects could manifest after 1 month.

RORα decreases oxidative stress through the induction of SOD2 and GPx1 expression and thereby protects against nonalcoholic steatohepatitis in mice.

AIMS: Increased hepatic oxidative stress and inflammation is the main cause of exacerbating nonalcoholic steatohepatitis (NASH). Retinoic acid-related orphan receptor α (RORα) regulates diverse target genes associated with lipid metabolism, and its expression level is low in the liver of patients with NASH. Here, we investigated the role of RORα in regulating hepatic oxidative stress and inflammation. RESULTS: First, cholesterol sulfate (CS), an agonist of RORα, lowered oxidative stress that was induced by 1.5 mM oleic acid in the primary cultures of hepatocytes. Second, exogenously introduced RORα or CS treatment induced the mRNA level of antioxidant enzymes, superoxide dismutase 2 (SOD2) and glutathione peroxidase 1 (GPx1), through the RORα response elements located in the upstream promoters of Sod2 and Gpx1. Third, RORα significantly decreased reactive oxygen species levels and mRNA levels of tumor necrosis factor α (TNFα) and interleukin-1ß that were induced by lipopolysaccharide or TNFα in Kupffer cells. Finally, the administration of JC1-40 decreased the signs of liver injury, lipid peroxidation, and inflammation in the MCD diet-induced NASH mice. INNOVATION AND CONCLUSION: We showed for the first time that RORα and its ligands protect NASH in mice by reducing hepatic oxidative stress and inflammation. Further, the molecular mechanism of the protective function of RORα against oxidative stress in the liver was revealed. These findings may offer a rationale for developing therapeutic strategies against NASH using RORα ligands.

Dynamic vaulting changes in V4c versus V4 posterior chamber phakic lenses under differing lighting conditions.

PURPOSE: To compare vaulting changes in eyes implanted with V4c and V4 implantable collamer lenses (ICLs) under differing lighting conditions. DESIGN: Noninterventional, cross-sectional comparative observational case series. METHODS: Fifty-six eyes of 38 patients implanted with V4c ICLs, and 54 eyes of 28 patients implanted with V4 ICLs were enrolled and analyzed. Anterior chamber depth, pupil size and postoperative vaulting were evaluated using a Visante optical coherence tomography system under photopic and mesopic conditions 1 month postoperatively. Refractive errors, keratometry values, axial lengths, intraocular pressures, anterior chamber volumes, and central corneal thicknesses were also recorded. RESULTS: No significant differences were noted in anterior chamber depth between photopic and mesopic conditions in either group. Significant decreases in vaulting and pupil size were detected under photopic conditions in both groups. Moreover, vaulting changes in eyes implanted with V4c ICLs were significantly larger than those in eyes implanted with V4 ICLs. CONCLUSIONS: V4c ICL vaulting decreased more prominently under photopic conditions than did V4 ICL vaulting. Therefore, postoperative vaulting under mesopic and photopic conditions should be considered when interpreting the vaulting of eyes implanted with V4c ICLs.

Changes of clinical manifestation of granular corneal deposits because of recurrent corneal erosion in granular corneal dystrophy types 1 and 2.

PURPOSE: To elucidate changes of clinical manifestation of granular corneal deposits after recurrent corneal erosion in granular corneal dystrophy types 1 and 2 (GCD1 and GCD2). METHODS: Six patients (5 patients with GCD2 and 1 with GCD1) were included. Slit-lamp photographs of all patients and Fourier domain optical coherence tomography images of 3 available patients were analyzed. In part II of this study, to evaluate deposit disappearance incidence (becoming annular granular deposits), we counted deposits of another 60 GCD2 heterozygotes according to decade of age as the following subgroups: discoid (filled-up) granular deposits, annular (ring-shaped) granular deposits, and lattice-like lesions. RESULTS: Granular deposits dropped off singularly during recurrent corneal erosion in GCD2 heterozygotes, becoming annular deposits. In contrast, confluent granular deposits in GCD2 homozygotes and GCD1 patients dropped off in groups. Reaccumulation was observed at the margin of the previously dropped off area in patients who were followed-up for an extended period. Part II of the study showed that annular deposits appeared in the third decade and increased gradually. Discoid granular deposits increased sharply in the 40s age group, and lattice-like lesions increased gradually from the third decade. CONCLUSIONS: The disappearance of granular deposits in GCD1 and GCD2 could be interpreted as evidence of drop-off phenomena with recurrent corneal erosion. With drop off of the deposits, various configurations and morphological changes of corneal deposits can occur in these corneal dystrophies.

Characteristic features of granular deposit formation in granular corneal dystrophy type 2.

PURPOSE: To describe the characteristic features of white granular deposits associated with granular corneal dystrophy type 2 (GCD2). METHODS: Five patients with GCD2 associated with the R124H mutation (2 homozygous GCD2 and 3 heterozygotes) were examined. The corneal deposits of all patients were assessed and reviewed by slit-lamp photographs. Density line profiles of corneal surfaces were evaluated around the discrete corneal deposits using Image J software. A Fourier-domain optical coherence tomography with cornea anterior module was used to visualize the characteristic surrounding features of these granular deposits. A histopathological study of the homozygous corneal specimen obtained after keratoplasty was performed. RESULTS: Slit-lamp images and densitometry line profiles showed that discrete granules were surrounded by relatively clear areas in all patients. The Fourier-domain optical coherence tomography image clearly showed highly reflective lesions, corresponding to the corneal deposits, surrounded by lower reflective areas. Histopathological study revealed comparable findings to the optical coherence tomography image. Serial comparison of slit-lamp photographs demonstrated a recurrence pattern after penetrating keratoplasty in a homozygous patient and natural progression in a heterozygote patient. They were similar in that diffuse fine granules gradually increased in density and discrete granular deposits also enlarged or were newly formed. CONCLUSIONS: In GCD2, discrete white granular deposits were surrounded by rather lucid areas and fine granular haze. These findings suggest that white granular deposits may be formed by aggregation of surrounding fine granules.