Albert S. Jun

Subnormal Cytokine Profile in the Tear Fluid of Keratoconus Patients

Keratoconus, historically viewed as a non-inflammatory disease, is an ectatic corneal disorder associated with progressive thinning of the corneal stroma. Recently, a few inflammatory mediators have been reported to be elevated in the tear fluid of keratoconus patients. Consequently, we investigated a wide range of inflammation regulating cytokines in the tears and sera of keratoconus and control subjects. Interleukin (IL)-1β, IL-4, IL-6, IL-10, IL-12, IL-13, IL-17, interferon (IFN)-γ, chemokine C-C motif ligand 5 (CCL5) and tumor necrosis factor (TNF)-α were tested in tear samples and sera of keratoconus and control individuals by multiplex immuno-bead assays. Selected cytokines were further tested by standard ELISA on pooled tear samples. All cytokines in the sera were generally low, with no significant changes between keratoconus and control subjects. However, in tear fluids, clear differences were detected between the two groups. These differences include increased IL-6, and decreased IL-12, TNF-α, IFN-γ, IL-4, IL-13 and CCL5 in keratoconus compared to control tear fluids. The decreases in IL-12, TNF-α and CCL5 were statistically significant, while the IL-13 decrease was statistically significant in the severe keratoconus group only. IL-17 could not be detected by multiplex immuno-bead assay, but showed an increase in keratoconus by conventional ELISA on a limited number of pooled tear samples. Our findings confirm increased IL-6, but dispute earlier reports of increased TNF-α, and suggest a cytokine imbalance in keratoconus disrupting corneal homeostasis. Moreover, an increase in IL-17 suggests tissue degenerative processes at work, contributing to the thinning and weakening of the corneal connective tissue in keratoconus.

Unfolded protein response in fuchs endothelial corneal dystrophy: a unifying pathogenic pathway?

PURPOSE To assess for activation of the unfolded protein response in corneal endothelium of Fuchs endothelial corneal dystrophy patients. DESIGN Retrospective, comparative case series of laboratory specimens. METHODS Corneal specimens of patients with Fuchs dystrophy and controls with corneal pathologic features other than Fuchs dystrophy were evaluated by transmission electron microscopy (TEM) to evaluate for structural changes of the rough endoplasmic reticulum in corneal endothelium. TEM images were evaluated for alterations of rough endoplasmic reticulum as a sign of unfolded protein response. Normal autopsy eyes, Fuchs dystrophy corneas, and keratoconus corneas were used for immunohistochemistry. Immunohistochemistry was performed on formalin-fixed, paraffin-embedded sections of patient corneas for 3 unfolded protein response markers (GRP78, the alpha subunit of eukaryotic initiation factor 2, C/EBP homologous protein) and 2 apoptosis markers (caspase 3 and 9). Immunohistochemistry signal quantitation of corneal endothelium for evaluation of marker expression was performed using automated software. Corneal sections were assessed quantitatively for levels of immunohistochemistry marker expression. RESULTS TEM showed enlargement of rough endoplasmic reticulum in corneal endothelium of all Fuchs dystrophy specimens. Immunohistochemistry quantitation demonstrated a significant increase in mean signal in corneal endothelium from Fuchs dystrophy patients for markers GRP78, the alpha subunit of eukaryotic initiation factor 2, C/EBP homologous protein, and caspase 9 compared with non-Fuchs dystrophy corneas (P < .05). CONCLUSIONS Results of both TEM and immunohistochemistry indicate activation of unfolded protein response in Fuchs dystrophy. Unfolded protein response activation leads to endothelial cell apoptosis in Fuchs dystrophy and may play a central pathogenic role in this disease.

An Alpha 2 Collagen VIII Transgenic Knock-in Mouse Model of Fuchs Endothelial Corneal Dystrophy Shows Early Endothelial Cell Unfolded Protein Response and Apoptosis

Fuchs endothelial corneal dystrophy (FECD) is a leading indication for corneal transplantation. FECD is characterized by progressive alterations in endothelial cell morphology, excrescences (guttae) and thickening of the endothelial basement membrane and cell death. Ultimately, these changes lead to corneal edema and vision loss. Due to the lack of vision loss in early disease stages and the decades long disease course, early pathophysiology in FECD is virtually unknown as studies of pathologic tissues have been limited to end-stage tissues obtained at transplant. The first genetic defect shown to cause FECD was a point mutation causing a glutamine to lysine substitution at amino acid position 455 (Q455K) in the alpha 2 collagen 8 gene (COL8A2) which results in an early onset form of the disease. Homozygous mutant knock-in mice with this mutation (Col8a2(Q455K/Q455K)) show features strikingly similar to human disease, including progressive alterations in endothelial cell morphology, cell loss and basement membrane guttae. Ultrastructural analysis shows the predominant defect as dilated endoplasmic reticulum (ER), suggesting ER stress and unfolded protein response (UPR) activation. Immunohistochemistry, western blotting, quantitative reverse transcriptase polymerase chain reaction and terminal deoxynucleotidyl transferase 2-deoxyuridine, 5-triphosphate nick end-labeling analyses support UPR activation and UPR-associated apoptosis in the Col8a2(Q455K/Q455K) mutant corneal endothelium. This study confirms the Q455K substitution in the COL8A2 gene as being sufficient to cause FECD in the first mouse model of this disease and supports the role of the UPR and UPR-associated apoptosis in the pathogenesis of FECD caused by COL8A2 mutations.

The keratoconus corneal proteome: Loss of epithelial integrity and stromal degeneration

UNLABELLED Keratoconus is a thinning corneal dystrophy that begins in the early teenage years and ultimately requires cornea transplantation to restore vision. Here we conducted a highly sensitive mass spectrometric analysis of the epithelium and the stroma from keratoconus and normal donor corneas. We identified a total of 932 and 1157 proteins in the consolidated data of the epithelium and stroma, respectively. Technical replicates showed strong correlations (≥0.88) in levels of all common proteins, indicating very low technical variations in the data. Analysis of the most increased (≥1.5 fold) and decreased (≤0.8 fold) proteins in the keratoconus corneal epithelial protein extracts identified proteins related to dermal diseases, inflammation, epithelial stratification and mesenchymal changes. Increased proteins included keratins 6A, 16 and vimentin, while the iron transporter lactotransferrin was decreased. The keratoconus stromal proteome suggests endoplasmic reticular stress, oxidative stress and widespread decreases in many extracellular matrix proteoglycan core proteins, lumican and keratocan, collagen types I, III, V and XII. Marked increase in apoptosis and endocytosis-related proteins suggest degenerative changes in keratocytes, the resident cells of the stroma. This is the most comprehensive proteome analysis of the cornea that highlights similarities of keratoconus with other neurodegenerative diseases. BIOLOGICAL SIGNIFICANCE This study provides, to our knowledge, the most comprehensive proteomic analysis of the vision threatening disease keratoconus, which affects a significant portion of the US and global populations. Using iTRAQ and LC/MS/MS, we have identified significant changes in the human corneal epithelium and stromal proteome that correlate to in vivo clinical findings. The protein changes identified will lead to molecular insights into disease pathogenesis and provide candidate genes for genetic studies of keratoconus.

A Clinicopathologic Series of Primary Graft Failure after Descemet's Stripping and Automated Endothelial Keratoplasty

PURPOSE To characterize the clinical and histologic features of primary graft failure after Descemets stripping and automated endothelial keratoplasty (DSAEK). DESIGN Retrospective observational case series. PARTICIPANTS Sixteen cases of DSAEK graft failure from 15 patients, all with detailed histologic examination of failed graft tissue. METHODS Hematoxylin-eosin, periodic acid-Schiff staining, and light microscopy were used to examine the failed DSAEK graft tissue from all patients. MAIN OUTCOME MEASURES Examination of specimens for corneal endothelial cell viability and host-donor interface characteristics. RESULTS Clinical history revealed that 88% (14/16) of studied DSAEK grafts detached before failure, and pathologic examination found that 75% (12/16) of failed grafts had atrophic corneal endothelium. Examples of residual host Descemets membrane in the graft site and improper donor trephination were also identified. CONCLUSIONS Marked loss of the corneal endothelium is the prominent feature of primary DSAEK graft failure. Examples of surgical features, such as incomplete Descemets stripping and residual full-thickness cornea with a DSAEK graft, are shown.

Direct Detection of Collagenous Proteins by Fluorescently Labeled Collagen Mimetic Peptides

Although fibrous collagens are major structural components of extracellular matrix in mammals, collagen overproduction is associated with many human diseases including cancers and fibrosis. Collagen is typically identified in biomedical research by Western blot and immunohistochemistry; however, anticollagen antibodies employed in these analyses are difficult to prepare and their affinities to collagen can diminish if collagen becomes denatured during analyses. Previously, we discovered that single-stranded collagen mimetic peptides [CMPs, sequence: (GlyProHyp)(9)] can bind to denatured collagen chains by triple helix hybridization. Here, we present collagen-specific staining methods using simple CMPs conjugated to common fluorophores (e.g., carboxyfluorescein), which allow direct detection of collagens and collagen-like proteins in SDS-PAGE and in various mammalian tissue sections. By directly staining SDS-PAGE gels with fluorescently labeled CMPs, both intact (type I, II, and IV) and MMP-1 cleaved collagen (type I) chains as well as complement factor C1q were detected. Collagen bands containing as little as 5 ng were optically visualized, while no staining was observed for fibronectin, laminin, and a collection of proteins from mammalian cell lysate. The CMP was unable to stain collagen-like bacterial protein, which contains numerous charged amino acids that are believed to stabilize triple helix in place of Hyp. We also show that fluorescently labeled CMPs can specifically visualize collagens in fixed tissue sections (e.g., skin, cornea, and bone) more effectively than anticollagen I antibody, and allow facile identification of pathologic conditions in fibrotic liver tissues.

Secondary angle closure caused by air migrating behind the pupil in descemet stripping endothelial keratoplasty.

Purpose: To report secondary angle closure caused by air migrating behind the pupil in the context of intraocular pressure (IOP) elevation in the early postoperative period after Descemet stripping endothelial keratoplasty (DSEK). Methods: A retrospective case series was conducted on 100 consecutive DSEK cases from 90 patients undergoing DSEK because of corneal disease from Fuchs corneal dystrophy, pseudophakic bullous keratopathy, aphakic bullous keratopathy, and iridocorneal endothelial syndrome. Preoperative and postoperative slit-lamp examinations and IOP measurements were ascertained for all 100 eyes. Main outcome measures included preoperative and postoperative IOP. Results: Thirteen of 100 eyes developed an IOP rise of greater than 30 mm Hg on the first postoperative day. Six of these 13 patients developed angle closure from air migrating posterior to the iris and causing iridocorneal adhesions. One of these 13 patients developed pupillary block from air anterior to iris. Six of 13 patients developed increased IOP without pupillary block or iridocorneal adhesions and had a history of preexisting primary or secondary glaucoma. Conclusions: A secondary angle closure associated with DSEK is reported with air migrating behind the iris, resulting in extensive iridocorneal adhesions. An acute increase in IOP after DSEK can also be induced by air anterior to the iris causing pupillary block. IOP spikes are much more common in the first few postoperative days after DSEK. Medical treatment can occasionally resolve air posterior to the iris, but if iridocorneal adhesions are extensive and persistent, air removal and angle reformation may be necessary.

Differential gene expression patterns of the developing and adult mouse cornea compared to the lens and tendon

The cornea continues to mature after birth to develop a fully functional, refractive and protective barrier tissue. Here we investigated the complex biological events underlying this process by profiling global genome-wide gene expression patterns of the immature postnatal day 10 and 7-week old adult mouse cornea. The lens and tendon were included in the study to increase the specificity of genes identified as upregulated in the corneal samples. Notable similarities in gene expression between the cornea and the tendon were in the mesenchymal extracellular matrix collagen (types I, III, V, VI) and proteoglycan (lumican, decorin and biglycan) genes. Expression similarities in the cornea and lens were limited to certain epithelial genes and the crystallins. Approximately 76 genes were over expressed in the cornea samples that showed basal expression levels in the lens and tendon. Thirty-two of these were novel with no known functions in the cornea. These include genes with a potential role in protection against oxidative stress (Dhcr24, Cdo1, Akr1b7, Prdx6), inflammation (Ltb4dh, Wdr1), ion transport (Pdzk1ip1, Slc12a2, Slc25a17) and transcription (Zfp36l3, Pdzk1ip1). Direct comparison of the cornea of two ages showed selective upregulation of 50 and 12 genes in the P10 and adult cornea, respectively. Of the upregulated P10 genes several encode extracellular matrix collagens and proteoglycans that are stable components of the adult cornea and their high transcriptional activity at P10 indicate a period of actie corneal growth and matrix deposition in the young cornea. Much less is known about the genes selectively over expressed in the adult cornea; some relate to immune response and innervations (Npy), and possibly to electron transport (Cyp24a1, Cyp2f2) and others of yet unknown functions in the cornea (Rgs10, Psmb8, Xlr4). This study detected expression of genes with known functions in the cornea, providing additional validation of the microarray experiments. Importantly, it identified several novel genes whose functions have not been investigated in the cornea.

Risk Factors for Eye Bank Preparation Failure of Descemet Membrane Endothelial Keratoplasty Tissue

PURPOSE To assess the results of a single eye bank preparing a high volume of Descemet membrane endothelial keratoplasty (DMEK) tissues using multiple technicians to provide an overview of the experience and to identify possible risk factors for DMEK preparation failure. DESIGN Cross-sectional study. METHODS setting: Lions VisionGift and Wilmer Eye Institute at Johns Hopkins Hospital. STUDY POPULATION All 563 corneal tissues processed by technicians at Lions VisionGift for DMEK between October 2011 and May 2014 inclusive. OBSERVATION PROCEDURES Tissues were divided into 2 groups: DMEK preparation success and DMEK preparation failure. MAIN OUTCOME MEASURES We compared donor characteristics, including past medical history. RESULTS The overall tissue preparation failure rate was 5.2%. Univariate analysis showed diabetes mellitus (P = .000028) and its duration (P = .023), hypertension (P = .021), and hyperlipidemia or obesity (P = .0004) were more common in the failure group. Multivariate analysis showed diabetes mellitus (P = .0001) and hyperlipidemia or obesity (P = .0142) were more common in the failure group. Elimination of tissues from donors either with diabetes or with hyperlipidemia or obesity reduced the failure rate from 5.2% to 2.2%. Trends toward lower failure rates occurring with increased technician experience also were found. CONCLUSIONS Our work showed that tissues from donors with diabetes mellitus (especially with longer disease duration) and hyperlipidemia or obesity were associated with higher failure rates in DMEK preparation. Elimination of tissues from donors either with diabetes mellitus or with hyperlipidemia or obesity reduced the failure rate. In addition, our data may provide useful initial guidelines and benchmark values for eye banks seeking to establish and maintain DMEK programs.

Descemet Stripping Automated Endothelial Keratoplasty After Failed Penetrating Keratoplasty: Survival, Rejection Risk, and Visual Outcome

IMPORTANCE Descemet stripping automated endothelial keratoplasty (DSAEK) for isolated endothelial dysfunction has become the preferred surgical option for many corneal surgeons. However, there are limited large-scale reports on DSAEK survival and clinical variables affecting the risk of rejection and failure after failed penetrating keratoplasty (PK). OBJECTIVE To report the survival, risk factors for graft rejection and failure, and visual outcome of DSAEK after failed PK. DESIGN, SETTING, AND PARTICIPANTS A multicenter retrospective interventional case series included patients recruited from 6 tertiary referral surgical centers: 3 in the United States, 2 in Europe, and 1 in Asia. A total of 246 consecutive eyes (246 patients) that underwent DSAEK after failed PK, with a minimum follow-up period of 1 month, was included. Data comprising demographic details, preoperative and postoperative risk factors, time to rejection, time to failure, and corrected distance visual acuity were collected. MAIN OUTCOMES AND MEASURES Cumulative probability of graft survival, hazard ratio estimates for survival, and corrected distance visual acuity were determined. RESULTS The mean (SD) recipient age was 63.2 (16.6) years and the median follow-up period was 17 months (interquartile range, 6-30 months). One-third of the grafts (n = 82) had follow-up data for more than 2 years; 18.3% had more than 1 failed PK before DSAEK. In total, 19.1% (47 of 246) of DSAEK grafts failed. The cumulative probability of DSAEK survival after a failed PK was 0.89 (95% CI, 0.84-0.92), 0.74 (95% CI, 0.64-0.81), and 0.47 (95% CI, 0.29-0.61) at 1 year, 3 years, and 5 years, respectively. Based on multivariate analysis, significant preoperative risk factors for failure were young recipient age (hazard ratio [HR], 5.18 [95% CI, 1.57-17.18]), previous tube filtration surgery (HR, 5.23 [95% CI, 1.47-7.33]), and rejection episodes before PK failure (HR, 3.28 [95% CI, 1.47-7.33]); single-surgeon centers had a protective effect. Any rejection episode prior to PK failure was a significant predictor of post-DSAEK rejection, which in turn was a significant predictor of DSAEK failure. After a median follow-up of 17 months, 33.3% of the grafts achieved 0.3 or greater logMAR (20/40) corrected distance visual acuity. CONCLUSIONS AND RELEVANCE Descemet stripping automated endothelial keratoplasty after failed PK combines greater wound stability and reduced suture-related complications, with visual outcomes and graft survival rates comparable to those of a second PK.