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PURPOSE: MicroRNAs (miRNAs) are a class of endogenous noncoding RNA and post transcriptionally modulate gene expression during development and disease. Our study investigated the differential miRNA expression in human Fuchs' endothelial corneal dystrophy (FECD) compared with normal endothelium to identify miRNA sequences that are involved in the pathogenesis of FECD. METHODS: Comparative miRNA expression profiles of endothelial samples obtained from FECD patients during lamellar corneal transplant surgery and from normal donor globes were generated using OpenArray plate technology. Differential expression of individual miRNAs was validated in the original and in independent samples using stem-loop RT qPCR assays. Expression of miRNA target genes was assessed using qPCR and tissue microarray (TMA) immunolabeling. RESULTS: Our results demonstrate downregulation of 87 miRNAs in FECD compared with normal endothelium (>3-fold change; P < 0.01). Correspondingly, DICER1, (encoding an endoribonuclease critical to miRNA biogenesis) showed a moderate but significant decrease in FECD samples (P < 0.05). Significant repression of three miR-29 family members (miR-29a-3p, miR-29b-2-5p, and miR-29c-5p) was paralleled by upregulation of their extracellular matrix associated mRNA targets collagen I and collagen IV. Tissue microarray immunolabeling showed histologically verifiable subendothelial collagen I and collagen IV deposition and increased endothelial laminin protein expression in FECD samples. CONCLUSIONS: The present study provides the first miRNA profile in FECD and normal endothelial cells and demonstrates widespread miRNA downregulation in FECD. Decreased endothelial expression of miR-29 family members may be associated with increased subendothelial extracellular matrix accumulation in FECD.

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PURPOSE: The aim was to produce 2 tissue microarrays (TMAs) for keratoconus (KC) corneas and to evaluate the expression of stress-related markers, epidermal growth factor receptor (EGFR), and 8-oxo-2'-deoxyguanosine (8-OHdG), in KC corneas. METHODS: The corneal buttons of 66 patients with KC were included in both TMAs; 10 Fuchs endothelial corneal dystrophy (FECD), 20 normal autopsy corneas, and 32 nonocular tissue cores served as controls. The expression of immunolabeling for EGFR and 8-OHdG in KC corneas was compared with those of the controls by TMAJ software using an H-score index. To further interpret our findings, pig eyes under different preservation conditions were stained for the same markers. RESULTS: With 2 TMAs, we designed an effective model to investigate KC corneas at the protein level. The EGFR in epithelial cells showed significant upregulation in KC specimens compared with that in FECD controls (P = 0.009), and this was also higher in autopsy controls compared with that in KC corneal samples (P = 0.0002). The 8-OHdG in epithelial cells was elevated in KC samples compared with that in the FECD specimens (P = 0.03), whereas autopsy controls showed higher levels compared with those shown by the KC corneal samples (P < 0.0001). Immunohistochemical staining intensities for both markers in pig corneas correlated with increased time to fixation. CONCLUSIONS: TMAs simultaneously enable efficient, high-throughput analysis of tissue samples. The upregulation of EGFR and 8-OHdG protein levels in KC epithelium compared with FECD controls implicates oxidative stress in KC corneas. The expression of these stress markers is increased depending on the time to preservation, which may explain the increased levels of these markers in autopsy control corneas.

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PURPOSE: To investigate the endothelial gene expression profile in a Col8a2 Q455K mutant knock-in mouse model of early-onset Fuchs' endothelial corneal dystrophy (FECD) and identify potential targets that can be correlated to human late-onset FECD. METHODS: Diseased or normal endothelial phenotypes were verified in 12-month-old homozygous Col8a2(Q455K/Q455K) mutant and wild-type mice by clinical confocal microscopy. An endothelial whole genome expression profile was generated by microarray-based analysis. Result validation was performed by real-time PCR. Endothelial COX2 and JUN expression was further studied in human late-onset FECD compared to normal samples. RESULTS: Microarray analysis demonstrated endothelial expression of 24,538 genes (162 up-regulated and 172 down-regulated targets) and identified affected gene ontology terms including Response to Stress, Protein Metabolic Process, Protein Folding, Regulation of Apoptosis, and Transporter Activity. Real-time PCR assessment confirmed increased Cox2 (P = 0.001) and Jun mRNA (P = 0.03) levels in Col8a2(Q455K/Q455K) mutant compared to wild-type mice. In human FECD samples, real-time PCR demonstrated a statistically significant increase in COX2 mRNA (P < 0.0001) and JUN mRNA (P = 0.002) and tissue microarray analysis showed increased endothelial COX2 (P = 0.02) and JUN protein (P = 0.04). CONCLUSIONS: The present study provides the first endothelial whole genome expression analysis in an animal model of FECD and represents a useful resource for future studies of the disease. In particular endothelial COX2 up-regulation warrants further investigation of its role in FECD.

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PURPOSE: To investigate the novel application of tissue microarray (TMA) technology to corneal disease and to report altered protein expression of senescence-associated cyclin-dependent kinase inhibitors p21 and p16 in Fuchs endothelial corneal dystrophy (FECD). METHODS: A TMA including 208 cores was generated from paraffin-embedded tissues, including corneal buttons of 50 FECD and 5 keratoconus patients retrieved after penetrating keratoplasty, 10 autopsy globes with nonpathologic corneas, and nonocular control specimens. TMA sections were immunolabeled for p21 and p16 and analyzed using a 9-grade scoring system (0-8). Result validation was performed by immunolabeling of individual whole tissue sections. Corneal endothelial p21 and p16 expression levels in FECD specimens compared with controls served as main outcome measures. RESULTS: TMA immunohistochemical analysis disclosed increased endothelial expression levels of nuclear p21 in FECD specimens (P < 0.05) and an altered endothelial p16 expression pattern. Immunolabeling of whole tissue sections showed statistically significant endothelial overexpression of both proteins (p21 and p16, P < 0.05). CONCLUSIONS: The present study introduces TMA technology as a valuable tool for molecular high-throughput profiling of corneal tissues. It demonstrates p21 and p16 overexpression in the corneal endothelium of genetically undifferentiated FECD patients supporting a role of cellular senescence in the pathogenesis of FECD.

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PURPOSE: To evaluate the onset of posterior vitreous detachment (PVD) including early changes at the vitreoretinal interface after uneventful phacoemulsification in nonmyopic eyes using optical coherence tomography (OCT) and ultrasound. DESIGN: Prospective consecutive study. METHODS: Patients undergoing cataract surgery at our unit between January and October 2010 were recruited and examined with OCT and ultrasound preoperatively. Inclusion criteria were complete vitreoretinal attachment, no ocular pathology other than cataract, and no previous ocular surgery. All patients underwent phacoemulsification with intraocular lens implantation. Postoperatively, OCT and ultrasound were performed 1 month and 3 months after surgery. Exclusion criteria were axial length≥25 mm, lattice degeneration, intraoperative complications, and incomplete follow-up. RESULTS: Forty-nine eyes of 49 patients could be enrolled in the study. Some degree of PVD was noted in 29 eyes (59.2%) 1 month after surgery and in 35 eyes (71.4%) 3 months after surgery. Moreover, a significant decrease in prevalence of initial PVD and a corresponding increase of more advanced PVD stages throughout the duration of the study was observed. In patients older than 70 years some degree of PVD was diagnosed in 92.3% compared to 47.8% in patients younger than 70 years (P=.002). CONCLUSION: OCT facilitates the detection of early vitreoretinal separation that indicates initial PVD. After phacoemulsification the prevalence of some degree of PVD is consequently more frequent when supplementary OCT is used. Furthermore, OCT discloses a significant progression of PVD in the postoperative course. Patients older than 70 years are more likely to develop pseudophakic PVD.

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PURPOSE: To investigate the early and late stages of posterior vitreous detachment (PVD) in the foveal area in correlation with age and gender. METHODS: Three hundred and thirty-five emmetropic eyes of 271 Caucasian patients (216 women/119 men) were examined by optical coherence tomography (OCT) and ultrasound (US). Eyes were classified into groups according to the patients age (up to 69.9; 70-74.9; 75-79.9; over 80 years) and to the clinical findings [Vitreous state: Detached in US; Detached in OCT; Foveal adhesion (FA); Attached vitreous]. RESULTS: The mean age was 76 ± 8 ranging from 44 to 89 years in female and 72 ± 10 ranging from 46 to 87 years in male subjects. The vitreous was attached in 32% of all eyes, 18.5% had FA, 18.5% were detached in OCT and 68% were detached in US. While prevalence of FA decreases with increasing age, OCT-diagnosed detachments did not change significantly with age. Between the ages of 70 and 75, an increase in PVD rates occurred. The prevalence of PVD was similar in both genders. Women were significantly older than men in the late-stage PVD in the eyes. CONCLUSION: The use of OCT and US enabled us to detect a partial or total PVD in 80% of the eyes. A sudden increase in late-stage PVD between the ages of 70 and 75 was observed, correlating with the reported age prevalence of various macular diseases. In contrast to myopics, both genders of elderly emmetropics have a similar prevalence of PVD.

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Cancer progression is often associated with the formation of malignant effusions. Vascular endothelial growth factor (VEGF) is a major regulator of vascular permeability and has been implicated as mediator of tumor progression. We examined the production and secretion of VEGF(165) in various primary cancer cells derived from malignant effusions, and the role of exogenous VEGF(165) as a mediator of effusion formation. VEGF(165) was constantly secreted by all cultured tumor cells in an mTOR-dependent manner, as it was inhibited by the mTOR inhibitor rapamycin. Secreted VEGF(165) showed functional activity by inducing endothelial leakiness and tumor cell-transendothelial migration in vitro, effects which could be reverted by the anti-VEGF antibody bevacizumab. Thus, mTOR inhibitors as well as bevacizumab should be considered as potential agents in cancer patients suffering from malignant effusions.

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