Martin Heur

Tear cathepsin S as a candidate biomarker for Sjögren's syndrome.

The diagnosis of Sjögrens syndrome (SS) in routine practice is largely a clinical one and requires a high index of suspicion by the treating physician. This great dependence on clinical judgment frequently leads to delayed diagnosis or misdiagnosis. Tear protein profiles have been proposed as simple and reliable biomarkers for the diagnosis of SS. Given that cathepsin S activity is increased in the lacrimal glands and tears of NOD mice (a murine model of SS), the aim of this study was to explore the clinical utility of using tear cathepsin S (CTSS) activity as a biomarker for SS.

Interleukin-1β-Induced Wnt5a Enhances Human Corneal Endothelial Cell Migration through Regulation of Cdc42 and RhoA

ABSTRACT Wnt5a can activate β-catenin-independent pathways for regulation of various cellular functions, such as migration, that play critical roles in wound repair. Investigation of Wnt5a signaling may help identify therapeutic targets for enhancing corneal endothelial wound healing that could provide an alternative to corneal transplantation in patients with blindness from endothelial dysfunction. However, Wnt5a signaling in corneal endothelial cells (CECs) has not been well characterized. In this study, we show transient induction of Wnt5a by interleukin-1β (IL-1β) stimulation proceeds through NF-κB in human CECs. This leads to binding of Fzd5 to Ror2, resulting in activation of disheveled protein (Dvl) and subsequently disheveled-associated activator of morphogenesis 1 (DAAM1). This leads to activation of Cdc42 and subsequent inhibition of RhoA. Inhibition of RhoA leads to parallel dephosphorylation and inactivation of LIM domain kinase 2 along with dephosphorylation and activation of slingshot 1, resulting in dephosphorylation and activation of cofilin and leading to enhanced cell migration. These findings suggest that Wnt5a enhances cell migration through activation of Cdc42 and inactivation of RhoA in human CECs.

Prosthetic Replacement of the Ocular Surface Ecosystem Scleral Lens Therapy for Patients With Ocular Symptoms of Chronic Stevens-Johnson Syndrome

PURPOSE To evaluate the results of Prosthetic Replacement of the Ocular Surface Ecosystem (PROSE) scleral lens treatment on visual acuity and function in patients with ocular symptoms of chronic Stevens-Johnson syndrome (SJS). DESIGN Retrospective interventional case series. METHODS setting: Single multi-specialty institutional practice. study population: A chart review from July 2009 to July 2013 identified 19 patients with ocular symptoms from chronic SJS who were referred for PROSE fitting evaluation. Three patients deemed appropriate candidates were excluded because they were lost to follow-up during the fitting process. Only 1 eye was fitted in 4 patients because anatomic changes prohibited PROSE fitting in the fellow eye. Another patient chose to have PROSE fitting only in 1 eye. A total of 27 eyes of 16 patients who completed PROSE fitting were included in this study. intervention: PROSE scleral lens fitting. outcome measures: Visual acuity and visual function were assessed before and after PROSE fitting using Snellen acuity and Ocular Surface Disease Index (OSDI) survey. The OSDI survey is a validated questionnaire that assesses ocular surface disease in the context of vision-related function, ocular symptoms, and environmental triggers. RESULTS Visual acuity improved from 0.43 ± 0.35 logMAR pre-PROSE to 0.14 ± 0.22 logMAR post-PROSE (P = .0007) in SJS patients. OSDI scores improved from 70.4 ± 19.0 pre-PROSE to 37.4 ± 23.2 post-PROSE (P = .0002) in the same cohort. CONCLUSION The results of this study show that PROSE treatment is a viable option for improving visual acuity and function in SJS patients who failed conventional treatment.

Lacritin-mediated regeneration of the corneal epithelia by protein polymer nanoparticles

The avascular corneal epithelium plays an important role in maintaining normal vision and protecting the corneal interior from environmental infections. Delayed recovery of ocular wounds caused by trauma or refractive surgery strengthens the need to accelerate corneal wound healing and better restore the ocular surface. To address this need, we fused elastin-like polypeptide (ELP) based nanoparticles SI with a model mitogenic protein called lacritin. Lacritin fused at the N-terminus of the SI diblock copolymer is called LSI. This LSI fusion protein undergoes thermo-responsive assembly of nanoparticles at physiologically relevant temperatures. In comparison to ELP nanoparticles without lacritin, LSI showed potent signs of lacritin specific effects on a human corneal epithelial cell line (HCE-T), which included enhancement of cellular uptake, calcium-mediated signaling, and closure of a scratch. In vivo, the corneas of non-obese diabetic mice (NOD) were found to be highly responsive to LSI. Fluorescein imaging and corneal histology suggested that topical administration of LSI onto the ocular surface significantly promoted corneal wound healing and epithelial integrity compared to mice treated with or without plain ELP. Most interestingly, it appears that ELP-mediated assembly of LSI is essential to produce this potent activity. This was confirmed by comparison to a control lacritin ELP fusion called LS96, which does not undergo thermally-mediated assembly at relevant temperatures. In summary, fusion of a mitogenic protein to ELP nanoparticles appears to be a promising new strategy to bioengineer more potent biopharmaceuticals with potential applications in corneal wound healing.

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

Interleukin (IL)‐1β 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‐1β signalling may help identify therapeutic targets for corneal wound healing; however, mechanisms such as cell migration, a component of IL‐1β‐induced wound healing response in human corneal endothelial cells (CEC), have not been well characterised.

Sources of Structural Autofluorescence in the Human Trabecular Meshwork

PURPOSE In situ 2-photon excitation fluorescence (TPEF) of the human trabecular meshwork (TM) reveals beams of heterogeneous autofluorescence (AF) comprising high intensity fluorescent fibers (AF-high) on a background of lower intensity fluorescence (AF-low). To determine the sources of this AF heterogeneity, we imaged human TM to characterize AF, second harmonic generation (SHG) for collagen, and eosin-labeled fluorescence identifying elastin. METHODS Corneoscleral rims retained after corneal transplantation were incubated with and without eosin, and imaged by TPEF. TPEF was collected through multiphoton bandpass filters to obtain AF, SHG (collagen bandwidth), and eosin-labeled fluorescence images. For qualitative comparisons, near-simultaneous image acquisition pairs of AF-SHG (+/-eosin coincubation), AF-eosin, and SHG-eosin were captured. For quantitative comparisons, multiple regions of interest (ROI) were defined in separate TM beam regions within the uveal and corneoscleral meshwork for image acquisition pairs of AF-SHG (without eosin coincubation) and SHG-eosin. We defined 18 ROI within each acquisition pair as the basis for Manders colocalization analysis. Perfect colocalization was defined as a Manders coefficient (Mcoeff) of 1. RESULTS Qualitatively and quantitatively, AF-low colocalized with SHG (Mcoeff=1), but not SHG signal-voids. AF-high colocalized with SHG signal-voids (Mcoeff=1), but not the SHG signal. Like AF-high, eosin-labeled fluorescence qualitatively and quantitatively colocalized (Mcoeff=1) with SHG signal-voids, but not the SHG signal. CONCLUSIONS Heterogeneous AF in human TM is comprised of high intensity signal originating from elastin fibers in beam cores and lower intensity signal originating from collagen. These findings are relevant to interpreting structural extracellular matrix signals in AF images of the TM.

Investigation of femtosecond laser-Enabled keratoplasty wound geometry using optical coherence tomography

Purpose: To measure the wound geometry after femtosecond laser-enabled keratoplasty (FLEK) using optical coherence tomography (OCT). Design: Prospective nonrandomized clinical study and laboratory study. Participants: Patients who were candidates for penetrating keratoplasty at an academic referral center. Methods: Wound architecture was measured and analyzed by OCT in 8 eyes of 8 consecutive patients who underwent FLEK. Femtosecond laser lamellar cuts were performed on 3 eye bank corneas, and the wound diameters were measured by OCT. Results: Sutures were completely removed on average at 1 month per decade of age after surgery. No cases of wound dehiscence were noted, and graft-host tissue apposition appeared excellent on OCT. The mean spectacle-corrected visual acuity 1 month after suture removal was 20/35, and mean astigmatism was 5.0 diopters by manifest refraction and 8.7 diopters by computerized topography. The measured graft diameter was smaller than the laser setting by 2.5% (P = 0.007). The cut diameters of the eye bank corneas were also slightly smaller than the laser setting. Conclusions: FLEK offers benefits of rapid wound healing and predictable wound geometry. The measured wound geometry agreed well with laser setting other than a small systematic deviation that could be explained by the mechanics of corneal applanation during the laser cut. OCT is a useful tool to measure the actual graft diameter after FLEK.

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.

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.

WNT10B Enhances Proliferation Through β-catenin and RAC1 GTPase in Human Corneal Endothelial Cells

Background: Adult human corneal endothelial cells are G1-arrested, often necessitating transplantation in patients with endothelial dysfunction. Results: WNT10B enhances proliferation in human corneal endothelial cells. Conclusion: Modulation of the WNT10B pathway could be used to treat vision loss because of corneal endothelial dysfunction. Significance: Modulation of the WNT10B pathway may provide a means of addressing the impending increase in donor cornea demand. 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 β-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 β-catenin-dependent and -independent pathways and suggest that its modulation could be used to treat vision loss secondary to corneal endothelial dysfunction.