Use of Bowman's membrane electrocautery in blind eyes with painful bullous keratopathy not amenable to corneal transplantation: a retrospective case series.

PURPOSE: This study evaluated the safety and efficacy of Bowman's membrane electrocautery in blind painful eyes with bullous keratopathy not amenable to corneal transplantation. METHODS: Eleven eyes of 11 subjects with painful bullous keratopathy and poor visual potential who underwent electrocautery of Bowman's membrane at a tertiary referral ophthalmology clinic were reviewed retrospectively. Subject demographics and preoperative and postoperative data were collected, including description of pain, slit lamp biomicroscopy, best corrected visual acuity, topical medication use, and complications. Efficacy of the procedure on pain reduction, bullae resolution, and topical medication use were assessed at post-operative visits. Safety was also evaluated based on any complications. RESULTS: Bowman's membrane electrocautery effectively resolved bullae in all eyes examined up to 6 months postoperatively; however, 2 eyes had recurrence by 1 year. Mean age at the time of surgery was 69.8 years and mean duration of follow-up was 15.4 months. Pain reduction was achieved in all eyes at 1 month, but 1 subject had pain recurrence by 6 months and another by 1 year. The median number of drops per day decreased from 6 preoperatively to 1.7 at 6 months. Two subjects who had underlying advanced ophthalmic disease had a mild reduction in vision. CONCLUSION: Bowman's membrane electrocautery is a safe and minimally invasive procedure for the management of painful bullous keratopathy in eyes with low vision potential and not amenable to corneal transplantation. Duration of effect appears to last at least 6 months and up to 3 years post-procedure.

Non-infectious and non-hereditary diseases of the corneal epithelium.

The corneal epithelium serves as a physical barrier and a refractive element. Therefore, diseases of the corneal epithelium can increase the risk for infection and causes vision loss. The corneal epithelium can be affected by a multitude of conditions, such as infections, hereditary diseases, depositions, trauma, autoimmune conditions, factitious disorders, and iatrogenic causes. Non-infectious and non-hereditary corneal epithelial diseases represent a collection of conditions with diverse etiologies and clinical presentations but similar patient symptoms. The differing therapeutic interventions for each condition make clinical distinction important. The clinical characteristics, disease course, pathophysiology and current treatments for non-infectious, non-hereditary corneal epithelial diseases are reviewed.

Tear Proteases and Protease Inhibitors: Potential Biomarkers and Disease Drivers in Ocular Surface Disease.

Tears are highly concentrated in proteins relative to other biofluids, and a notable fraction of tear proteins are proteases and protease inhibitors. These components are present in a delicate equilibrium that maintains ocular surface homeostasis in response to physiological and temporal cues. Dysregulation of the activity of protease and protease inhibitors in tears occurs in ocular surface diseases including dry eye and infection, and ocular surface conditions including wound healing after refractive surgery and contact lens (CL) wear. Measurement of these changes can provide general information regarding ocular surface health and, increasingly, has the potential to give specific clues regarding disease diagnosis and guidance for treatment. Here, we review three major categories of tear proteases (matrix metalloproteinases, cathepsins, and plasminogen activators [PAs]) and their endogenous inhibitors (tissue inhibitors of metalloproteinases, cystatins, and PA inhibitors), and the changes in these factors associated with dry eye, infection and allergy, refractive surgery, and CLs. We highlight suggestions for development of these and other protease/protease inhibitor biomarkers in this promising field.

Fibroblast growth factor 2 induces proliferation and fibrosis via SNAI1-mediated activation of CDK2 and ZEB1 in corneal endothelium.

Investigating stimulation of endogenous wound healing in corneal endothelial cells (CECs) may help address the global shortage of donor corneas by decreasing the number of transplants performed for blindness because of endothelial dysfunction. We previously reported that IL-1ß stimulation leads to fibroblast growth factor (FGF2) expression, enhancing migration and proliferation of mammalian CECs. However, FGF2 also promotes the endothelial-mesenchymal transition, which can lead to retrocorneal membrane formation and blindness. This prompted us to investigate downstream FGF2 signaling targets that could be manipulated to prevent retrocorneal membrane formation. FGF2 stimulation altered cell morphology and induced expression of mesenchymal transition marker genes such as snail family transcriptional repressor 1 (SNAI1), SNAI2, zinc finger E-box-binding homeobox 1 (ZEB1), and ZEB2 This, in turn, induced expression of fibronectin, vimentin, and type I collagen, and suppressed E-cadherin in CECs in vitro and ex vivo siRNA-mediated SNAI1 knockdown revealed that SNAI1 induces ZEB1 expression, in turn inducing expression of type I collagen, the major component of retrocorneal membranes, and of cyclin-dependent kinase 2 (CDK2) and cyclin E1, promoting cell proliferation. siRNA-mediated knockdown of SNAI1 or ZEB1, but not of CDK2, inhibited FGF2-dependent expression of fibronectin, vimentin, and type I collagen and of suppression of E-cadherin expression. We conclude that SNAI1 is a key regulator of FGF2-dependent mesenchymal transition in human ex vivo corneal endothelium, with ZEB1 regulating type I collagen expression and CDK2 regulating cell proliferation. These results suggest that SNAI1 promotes fibrosis and cell proliferation in human corneal endothelium through ZEB1 and CDK2.

Injury induces endothelial to mesenchymal transition in the mouse corneal endothelium in vivo via FGF2.

Purpose: To determine whether the mouse corneal endothelium enters endothelial to mesenchymal transition (EndoMT) following surgical injury in vivo. Methods: The corneal endothelium in anesthetized mice was surgically injured in vivo under direct visualization. The secretion of interleukin-1 beta (IL-1ß) and fibroblast growth factor 2 (FGF2) into the aqueous humor was analyzed with western blotting. The expression of FGF2, Snai1, Zeb1, Col1a1, Col1a2, Fn1, Vim, Cdk2, Ccne1, and Cdh1 was analyzed with semiquantitative RT-PCR in the mouse corneal endothelium ex vivo and in vivo. Knockdown of FGF2 was done using siRNA. Col8a2 was used as a corneal endothelial marker, and Keratocan (Ktcn) was used as a stromal marker. ß-actin was used as a loading control. Results: Sequential expression of IL-1ß and FGF2 was detected in the aqueous humor after surgical injury. FGF2 treatment induced expression of endothelial to mesenchymal transition-related genes including Snai1, and Zeb1 in the mouse ex vivo corneal endothelium. This led to increased expression of Col1a1, Col1a2, Fn1, and Vim and suppression of Cdh1 in a time-dependent manner. Expression of FGF2, Snai1, Zeb1, Col1a1, Col1a2, Fn1, Vim, Cdk2, and Ccne1 was completely abolished by FGF2 siRNA knockdown in the mouse corneal endothelium ex vivo. Surgical injury induced FGF2 expression in the in vivo mouse corneal endothelium. The injury-dependent expression of FGF2, Snai1, Zeb1, Col1a1, Col1a2, Fn1, Vim, Cdk2, and Ccne1 and the suppression of Cdh1 were inhibited by siRNA knockdown of FGF in the mouse corneal endothelium in vivo. Moreover, siRNA knockdown of FGF2 inhibited the formation of the injury-dependent retrocorneal membrane in the in vivo mouse corneal endothelium. Conclusions: These findings suggest that after surgical injury, FGF2 induces the expression of EndoMT-related genes Snai1, Zeb1, Col1a1, Col1a2, Fn1, Vim, Cdk2, and Ccne1 in the mouse corneal endothelium in vivo, similar to the human corneal endothelium ex vivo.

Prosthetic Replacement of the Ocular Surface Ecosystem Scleral Lens Therapy for Exposure Keratopathy.

OBJECTIVES: To investigate the utility of Prosthetic Replacement of the Ocular Surface Ecosystem (PROSE) scleral lenses in patients with exposure keratopathy, with outcomes based on changes in visual acuity, visual function, and corneal staining. METHODS: A retrospective interventional case series of patients evaluated and treated from September 2009 through June 2014 at a single multi-specialty institutional practice. Eighteen of 29 patients with exposure keratoconjunctivitis, lagophthalmos, ectropion, or lid retraction, referred to USC Eye Institute after failing conventional therapies completed PROSE scleral lens fitting and were included in the study. Visual function was assessed before and after PROSE fitting with the Ocular Surface Disease Index (OSDI) survey. Visual acuity (VA) and corneal staining changes were also evaluated before and after treatment. RESULTS: Visual acuity improved from 0.60±0.68 logMAR pre-PROSE to 0.25±0.34 logMAR (Z=-3.81, P=0.00014) post-PROSE, which corresponds to an improvement of about 20/80 to 20/35 on Snellen VA. Ocular Surface Disease Index scores improved from 56.54±29.75 pre-PROSE to 24.98±21.23 post-PROSE (Z=-2.98, P=0.0029), and corneal staining values decreased from 2.17±0.84 pre-PROSE to 0.64±0.70 post-PROSE (Z=-3.27, P=0.011). CONCLUSIONS: The results suggest that PROSE scleral lens therapy is effective in patients with exposure keratopathy who had failed conventional therapies and can serve as an alternative to lid surgery.

WNT10B enhances proliferation through ß-catenin and RAC1 GTPase in human corneal endothelial cells.

The cornea is the anterior, transparent tissue of the human eye that serves as its main refractive element. Corneal endothelial cells are arranged as a monolayer on the posterior surface of the cornea and function as a pump to counteract the leakiness of its basement membrane. Maintaining the cornea in a slightly dehydrated state is critical for the maintenance of corneal transparency. Adult human corneal endothelial cells are G1-arrested, even in response to injury, leading to an age-dependent decline in endothelial cell density. Corneal edema and subsequent vision loss ensues when endothelial cell density decreases below a critical threshold. Vision loss secondary to corneal endothelial dysfunction is a common indication for transplantation in developed nations. An impending increase in demand for and a current global shortage of donor corneas will necessitate the development of treatments for vision loss because of endothelial dysfunction that do not rely on donor corneas. Wnt ligands regulate many critical cellular functions, such as proliferation, making them attractive candidates for modulation in corneal endothelial dysfunction. We show that WNT10B causes nuclear transport and binding of RAC1 and ß-catenin in human corneal endothelial cells, leading to the activation of Cyclin D1 expression and proliferation. Our findings indicate that WNT10B promotes proliferation in human corneal endothelial cells by simultaneously utilizing both ß-catenin-dependent and -independent pathways and suggest that its modulation could be used to treat vision loss secondary to corneal endothelial dysfunction.

Interleukin-1ß enhances cell migration through AP-1 and NF-κB pathway-dependent FGF2 expression in human corneal endothelial cells.

BACKGROUND INFORMATION: Interleukin (IL)-1ß is a major pro-inflammatory cytokine that plays a crucial role in the regulation of inflammation and wound healing in the cornea. Elucidation of IL-1ß signalling may help identify therapeutic targets for corneal wound healing; however, mechanisms such as cell migration, a component of IL-1ß-induced wound healing response in human corneal endothelial cells (CEC), have not been well characterised. RESULTS: Stimulation of human CEC with IL-1ß activated expression of fibroblast growth factor 2 (FGF2) and resulted in enhanced cell migration. This, in turn, was abolished by treatment with either IL-1 receptor antagonist or SU-5402, a pan-fibroblast growth factor signalling inhibitor. Phosphatidyl inositol (PI) 3-kinase or IL receptor-associated kinase 1/4 antagonists demonstrated that IL receptor-associated kinase 1/4 activates PI 3-kinase, which in turn phosphorylates p38 and inhibitor κB kinase α/ß, leading to FGF2 expression through activation of activator protein 1 (AP-1) and nuclear factor kappa-light-chain enhancer of activated B cells (NF-κB) in human CEC. Treatment of IL-1ß-stimulated human CEC with either AP-1 or NF-κB antagonists decreased FGF2 expression and resulted in reduced IL-1ß-enhanced cell migration. Co-treatment of IL-1ß-stimulated human CEC with both inhibitors completely blocked FGF2 expression and IL-1ß-enhanced cell migration. Chromatin immunoprecipitation assays demonstrated that AP-1 and NF-κB directly bind to the FGF2 promoter following IL-1ß stimulation. CONCLUSIONS: The results show that binding of IL-1ß to its receptor in human CEC leads to parallel activation of AP-1 and NF-κB pathways, leading, in turn, to FGF2 expression and enhanced cell migration.

Regenerative potential of the zebrafish corneal endothelium.

Corneal transparency, critical for clear vision, is maintained in part by the pump function of the corneal endothelial cells that are arrested in G(1) phase of the cell cycle in adult humans. Thus loss of endothelial cells leads to a decrease in endothelial cell density. A decrease below a critical threshold results in corneal edema and subsequent vision loss. Corneal edema due to endothelial dysfunction is a common indication for transplantation in developed countries. The zebrafish has emerged as a model for vertebrate regeneration due to its ease of genetic manipulation and remarkable regenerative capacity. The purpose of this study was to investigate the response and regenerative potential of the zebrafish corneal endothelium to pharmacological and mechanical injury. Similar to the human cornea, Na(+)/K(+) ATPase activity is necessary to maintain the pump function as intracameral injection of ouabain resulted in an increase in central corneal thickness. Surgical removal of the majority of the central corneal endothelium resulted in a similar increase in corneal thickness. Remarkably, by just one week post-injury the central corneal endothelium had largely re-formed. Immunofluorescence of phosphorylated histone H3 indicated that this recovery correlated with corneal endothelial cells re-entering the cell cycle. In conclusion, our results establish zebrafish as a useful model of corneal injury and repair that may offer insights into the mechanism of cell cycle arrest in human corneal endothelial cells.

Spatiotemporal gene targeting in the mouse corneal endothelium.

PURPOSE: The inducible Cre-ERT2 recombinase system allows for temporal control of gene targeting, and it is useful to studying adult function of genes that have critical developmental roles. The Zeb1flox/flox: UBC-CreERT2 mouse was generated to conditionally target Zeb1 to investigate its role in mesenchymal transition in the mouse corneal endothelium in vivo. MATERIALS AND METHODS: Hemizygous UBC-CreERT2 mice were crossed with homozygous mice harboring loxP-flanked Zeb1 alleles (Zeb1flox/flox) to generate the Zeb1flox/flox: UBC-CreERT2 mouse. 4-hydroxytamoxifen (4-OHT) exposure leads to excision of exon 6 of Zeb1, resulting in a loss function allele in the Zeb1flox/flox: UBC-CreERT2 mouse. Intracameral 4-OHT injection further isolates Zeb1 targeting to the anterior chamber. Mesenchymal transition and induction of Zeb1 expression in the corneal endothelium was achieved using FGF2 in ex vivo organ culture. Gene expression was analyzed by semi-quantitative reverse transcription-polymerase chain reaction and by immunoblotting in the mouse corneal endothelium in vivo. RESULTS: Following Cre-mediated targeting of Zeb1 by intracameral 4-OHT injection in Zeb1flox/flox: UBC-CreERT2 mice, FGF2 treatment in ex vivo organ culture resulted in abrogation of Zeb1 mRNA and protein expression in the corneal endothelium. CONCLUSION: The data show Zeb1, a critical mediator of fibrosis in corneal endothelial mesenchymal transition, can be targeted by intracameral injection of 4-OHT in the mouse corneal endothelium in vivo. These results suggest that genes with critical developmental roles can be targeted at a specific time in the corneal endothelium to study its role in adult disease using an inducible Cre-Lox strategy.

ZEB1 Mediates Fibrosis in Corneal Endothelial Mesenchymal Transition Through SP1 and SP3.

Purpose: ZEB1 is induced during endothelial-mesenchymal transition (EnMT) in the cornea. Induction of SP1 and SP3 by ZEB1 along with identification of putative SP1 and SP3 binding sites in promoters of EnMT-associated gene lead us to investigate their roles in retrocorneal membrane formation in the corneal endothelium. Methods: Expressions of SP1, SP3, and EnMT associated genes were analyzed by immunoblotting and semiquantitative reverse transcription polymerase chain reaction. Accell SMARTpool siRNAs targeting ZEB1, SP1, and SP3 were used for gene knockdown. SP1 and SP3 binding to promoters of EnMT associated genes was investigated by chromatin immunoprecipitation assay. Corneal endothelium in mice was surgically injured in vivo under direct visualization. Results: Transient Fibroblast Growth Factor 2 stimulation increased the expression of both SP1 and SP3 in the human corneal endothelium ex vivo. ZEB1 siRNA knockdown inhibited FGF2-induced SP1 mRNA and protein but not the expression of SP3. FGF2-induced expression of EnMT-related genes, such as fibronectin, vimentin, and type I collagen, was reduced by both SP1 and SP3 siRNA knockdown, with inhibition of SP1 having a greater inhibitory effect than SP3. Additionally, although SP1 and SP3 proteins were found to bind together, SP1 and SP3 could bind to the same promoter binding sites of EnMT-related genes in the absence of the other. Moreover, siRNA knockdown of Zeb1 inhibited injury-dependent RCM formation in mouse corneal endothelium in vivo. Conclusions: Zeb1, through SP1 and SP3, plays a central role in mesenchymal transition induced fibrosis in the corneal endothelium and suggests that Zeb1 could be targeted to inhibit anterior segment fibrosis.

Organogenesis and distribution of the ocular lymphatic vessels in the anterior eye.

Glaucoma surgeries, such as trabeculectomy, are performed to lower intraocular pressure to reduce risk of vision loss. These surgeries create a new passage in the eye that reroutes the aqueous humor outflow to the subconjunctival space, where the fluid is presumably absorbed by the conjunctival lymphatics. Here, we characterized the development and function of the ocular lymphatics using transgenic lymphatic reporter mice and rats. We found that the limbal and conjunctival lymphatic networks are progressively formed from a primary lymphatic vessel that grows from the nasal-side medial canthus region at birth. This primary lymphatic vessel immediately branches out, invades the limbus and conjunctiva, and bidirectionally encircles the cornea. As a result, the distribution of the ocular lymphatics is significantly polarized toward the nasal side, and the limbal lymphatics are directly connected to the conjunctival lymphatics. New lymphatic sprouts are produced mainly from the nasal-side limbal lymphatics, posing the nasal side of the eye as more responsive to fluid drainage and inflammatory stimuli. Consistent with this polarized distribution of the ocular lymphatics, a higher drainage efficiency was observed in the nasal side than the temporal side of the eye when injected with a fluorescent tracer. In contrast, blood vessels are evenly distributed at the anterior surface of the eyes. Also, we found that these distinct vascular distribution patterns were conserved in human eyes. Together, our study demonstrated that the ocular surface lymphatics are more densely present in the nasal side and uncovered the potential clinical benefits in selecting the nasal side as a glaucoma surgery site to improve fluid drainage.

Expression of interleukin-1 receptor antagonist in human cornea.

The purpose of this study was to confirm the expression of interleukin-1 receptor antagonist (IL-1 Ra) in the human cornea. Four samples of human ex vivo corneal epithelium were obtained from patients undergoing photorefractive keratectomy. RT-PCR was performed using mRNA isolated from the corneal epithelium and oligo-dT primers. PCR was performed on the cDNA products using primers specific for human IL-1 Ra. The PCR products were subcloned and sequenced. Human cornea sections were prepared from eyes enucleated for choroidal melanoma. Immunocytochemistry was performed using goat anti-mouse polyclonal IL-1 Ra IgG and NL-577 conjugated donkey anti-goat IgG. IL-1 Ra mRNA was expressed in all ex vivo corneal epithelium samples as confirmed by sequencing of the PCR products. Immunofluorescence studies revealed strongest expression of IL-1 Ra in the superficial apical layer of corneal epithelium. Expression of IL-1 Ra may represent an endogenous mechanism of down-regulating the effects of epithelial- and tear-derived IL-1alpha and IL-1beta on the intact epithelium in the unwounded cornea and stromal cells after injury.

Complete ring cyst of iris pigment epithelium documented by ultrasound biomicroscopy.

The authors report a complete ring cyst of the iris pigment epithelium confirmed by ultrasound biomicroscopy. In this case, ultrasound biomicroscopy effectively excluded the differential diagnoses of ring melanoma of the iris and multiple separate cysts of the iris pigment epithelium.

Ultrasonic Microelastography to Assess Biomechanical Properties of the Cornea.

OBJECTIVE: To both qualitatively and quantitatively investigate corneal biomechanical properties through an ultrasonic microelastography imaging system, which is potentially useful in the diagnosis of diseases, such as keratoconus, postrefractive keratectasia, and tracking treatment such as cross-linking surgery. METHODS: Our imaging system has a dual-frequency configuration, including a 4.5 MHz ring transducer to push the tissue and a confocally aligned 40 MHz needle transducer to track micron-level displacement. Two-dimensional/three-dimensional acoustic radiation force impulse (ARFI) imaging and Young's modulus in the region of interest were performed on ex vivo porcine corneas that were either cross-linked using formalin solution or preloaded with intraocular pressure (IOPs) from 5 to 30 mmHg. RESULTS: The increase of corneal stiffness and the change in cross-linked volume following formalin crosslinking could be precisely observed in the ARFI images and reflected by the reconstructed Young's modulus while the B-mode structural images remained almost unchanged. In addition, the relationship between the stiffness of the cornea and IOPs was investigated among 12 porcine corneas. The corneal stiffness is significantly different at various IOPs and has a tendency to become stiffer with increasing IOP. CONCLUSION: Our results demonstrate the principle of using ultrasonic microelastography techniques to image the biomechanical properties of the cornea. Integrating high-resolution ARFI imaging labeled with reconstructed Young's modulus and structural imaging of the cornea can potentially lead to a routinely performed imaging modality in the field of ophthalmology.

The value of keratometry and central corneal thickness measurements in the clinical diagnosis of Marfan syndrome.

PURPOSE: To explore the utility of keratometry and central corneal thickness (CCT) measurements in diagnosis of Marfan syndrome. DESIGN: Retrospective chart review of 211 patients referred for ocular examination to rule out Marfan syndrome. METHODS: Patients were categorized into Marfan or control groups using current clinical diagnostic criteria. Patients in whom Marfan syndrome could be neither diagnosed nor ruled out were categorized as inconclusive. Those with other connective tissue diseases were excluded from the study. T tests were performed on keratometry and CCT values, and odds ratios (ORs) were calculated. RESULTS: Sixty-two patients were in the Marfan group and 98 patients were in the control group. Mean age of the Marfan group was 22.3 years vs 19.3 years in the control group (P = .23). Marfan patients had significantly lower keratometry and CCT values than controls (40.8 diopters (D) [Marfan] vs 43.3 D [control], P = 4.0 x 10(-19); and 543.5 microm [Marfan] vs 564.2 microm [control], P = 9.1 x 10(-6)). The maximum OR for keratometry (17.6) was calculated at 42 D. In Marfan syndrome patients without ectopia lentis, these values were 41.5 D (P = .00026 vs control) and 542.0 microm (P = .0011 vs control). CONCLUSION: There was a highly significant difference in keratometry values between Marfan and control patients, and values less than 42 D could be used as a clinical diagnostic criterion for Marfan syndrome. Significant overlap in CCT values between Marfan and control patients suggests that further investigation is necessary to determine the clinical utility of CCT measurements.

Clusterin from human clinical tear samples: Positive correlation between tear concentration and Schirmer strip test results.

PURPOSE: To investigate the relationship between tear concentration of the homeostatic protein clusterin (CLU) and dry eye signs and symptoms, and to characterize tear CLU protein. METHODS: Two independent studies were conducted, one in Tucson (44 subjects), the other in Los Angeles (52 subjects). A cohort study design was employed to enroll patients without regard to dry eye diagnosis. Dry eye signs and symptoms were assessed using clinical tests. Tear samples were collected by Schirmer strip, and also by micropipette at slit lamp when possible. CLU from both sample types was quantified by immunoassay. The relationship between CLU concentration and clinical test scores was determined by Pearson's correlation coefficient (for individual eyes) and multiple linear regression analysis (including both eyes). CLU was also evaluated biochemically by western blotting. RESULTS: In the Tucson cohort, a positive correlation was observed between tear CLU concentration and results of the Schirmer strip test, a measure of tear flow (p = 0.021 includes both eyes). This result was corroborated in the Los Angeles cohort (p = 0.013). The mean tear CLU concentration was 31 ±â€¯14 µg/mL (n = 18 subjects, 33 eyes; range = 7-48 µg/mL). CLU from clinical tear samples appeared biochemically similar to CLU from a non-clinical tear sample and from blood plasma. CONCLUSIONS: Results support the hypothesis that an optimal concentration of tear CLU is important for ocular surface health, and that this drops below the effective threshold in dry eye. Tear CLU measurement might identify patients that could benefit from supplementation. Information about concentration will aid development of therapeutic dosage parameters.

Increased Cathepsin S activity associated with decreased protease inhibitory capacity contributes to altered tear proteins in Sjögren's Syndrome patients.

Cathepsin S (CTSS) activity is elevated in Sjögren's Syndrome (SS) patient tears. Here we tested whether protease inhibition and cystatin C (Cys C) levels are reduced in SS tears, which could lead to enhanced CTSS-driven degradation of tear proteins. CTSS activity against Cys C, LF and sIgA was tested in SS or healthy control tears. Tears from 156 female subjects (33, SS; 33, rheumatoid arthritis; 31, other autoimmune diseases; 35, non-autoimmune dry eye (DE); 24, healthy controls) were analyzed for CTSS activity and Cys C, LF, and sIgA levels. Cys C and LF showed enhanced degradation in SS tears supplemented with recombinant CTSS, but not supplemented healthy control tears. CTSS activity was significantly increased, while Cys C, LF and sIgA levels were significantly decreased, in SS tears compared to other groups. While tear CTSS activity remained the strongest discriminator of SS in autoimmune populations, combining LF and CTSS improved discrimination of SS beyond CTSS in DE patients. Reductions in Cys C and other endogenous proteases may enhance CTSS activity in SS tears. Tear CTSS activity is reconfirmed as a putative biomarker of SS in an independent patient cohort while combined LF and CTSS measurements may distinguish SS from DE patients.

Clusterin Seals the Ocular Surface Barrier in Mouse Dry Eye.

Dry eye is a common disorder caused by inadequate hydration of the ocular surface that results in disruption of barrier function. The homeostatic protein clusterin (CLU) is prominent at fluid-tissue interfaces throughout the body. CLU levels are reduced at the ocular surface in human inflammatory disorders that manifest as severe dry eye, as well as in a preclinical mouse model for desiccating stress that mimics dry eye. Using this mouse model, we show here that CLU prevents and ameliorates ocular surface barrier disruption by a remarkable sealing mechanism dependent on attainment of a critical all-or-none concentration. When the CLU level drops below the critical all-or-none threshold, the barrier becomes vulnerable to desiccating stress. CLU binds selectively to the ocular surface subjected to desiccating stress in vivo, and in vitro to the galectin LGALS3, a key barrier component. Positioned in this way, CLU not only physically seals the ocular surface barrier, but it also protects the barrier cells and prevents further damage to barrier structure. These findings define a fundamentally new mechanism for ocular surface protection and suggest CLU as a biotherapeutic for dry eye.