Satoshi Kawasaki

LRIG1 inhibits STAT3-dependent inflammation to maintain corneal homeostasis.

Corneal integrity and transparency are indispensable for good vision. Cornea homeostasis is entirely dependent upon corneal stem cells, which are required for complex wound-healing processes that restore corneal integrity following epithelial damage. Here, we found that leucine-rich repeats and immunoglobulin-like domains 1 (LRIG1) is highly expressed in the human holoclone-type corneal epithelial stem cell population and sporadically expressed in the basal cells of ocular-surface epithelium. In murine models, LRIG1 regulated corneal epithelial cell fate during wound repair. Deletion of Lrig1 resulted in impaired stem cell recruitment following injury and promoted a cell-fate switch from transparent epithelium to keratinized skin-like epidermis, which led to corneal blindness. In addition, we determined that LRIG1 is a negative regulator of the STAT3-dependent inflammatory pathway. Inhibition of STAT3 in corneas of Lrig1-/- mice rescued pathological phenotypes and prevented corneal opacity. Additionally, transgenic mice that expressed a constitutively active form of STAT3 in the corneal epithelium had abnormal features, including corneal plaques and neovascularization similar to that found in Lrig1-/- mice. Bone marrow chimera experiments indicated that LRIG1 also coordinates the function of bone marrow-derived inflammatory cells. Together, our data indicate that LRIG1 orchestrates corneal-tissue transparency and cell fate during repair, and identify LRIG1 as a key regulator of tissue homeostasis.

Ocular forward light scattering and corneal backward light scattering in patients with dry eye.

PURPOSEnTo evaluate ocular forward light scattering and corneal backward light scattering in patients with dry eye.nnnMETHODSnThirty-five eyes in 35 patients with dry eye and 20 eyes of 20 healthy control subjects were enrolled. The 35 dry eyes were classified into two groups according to whether superficial punctate keratopathy in the central 6-mm corneal zone (cSPK) was present or not. Ocular forward light scattering was quantified with a straylight meter. Corneal backward light scattering from the anterior, middle, and posterior corneal parts was assessed with a corneal densitometry program using the Scheimpflug imaging system.nnnRESULTSnBoth dry eye groups had significantly higher intraocular forward light scattering than the control group (both P<0.05). The dry eye group with cSPK had significantly higher values in anterior and total corneal backward light scattering than the other two groups. Moderate positive correlations were observed between the cSPK score and corneal backward light scattering from the anterior cornea (R=0.60, P<0.001) and corneal backward light scattering from the total cornea (R=0.54, P<0.001); however, no correlation was found between cSPK score and ocular forward light scattering (R=0.01, P=0.932).nnnCONCLUSIONSnOcular forward light scattering and corneal backward light scattering from the anterior cornea were greater in dry eyes than in normal eyes. Increased corneal backward light scattering in dry eye at least partially results from cSPK overlying the optical zone.

OVOL2 Maintains the Transcriptional Program of Human Corneal Epithelium by Suppressing Epithelial-to-Mesenchymal Transition

In development, embryonic ectoderm differentiates into neuroectoderm and surface ectoderm using poorly understood mechanisms. Here, we show that the transcription factor OVOL2 maintains the transcriptional program of human corneal epithelium cells (CECs), a derivative of the surface ectoderm, and that OVOL2 may regulate the differential transcriptional programs of the two lineages. A functional screen identified OVOL2 as a repressor of mesenchymal genes to maintain CECs. Transduction of OVOL2 with several other transcription factors induced the transcriptional program of CECs in fibroblasts. Moreover, neuroectoderm derivatives were found to express mesenchymal genes, and OVOL2 alone could induce the transcriptional program of CECs in neural progenitors by repressing these genes while activating epithelial genes. Our data suggest that the difference between the transcriptional programs of some neuroectoderm- and surface ectoderm-derivative cells may be regulated in part by a reciprocallyxa0repressive mechanism between epithelial and mesenchymal genes, as seen in epithelial-to-mesenchymal transition.

Clinical Outcomes of Phototherapeutic Keratectomy in Eyes With Thiel-Behnke Corneal Dystrophy

PURPOSEnTo investigate the functional and morphologic midterm outcome of phototherapeutic keratectomy (PTK) for Thiel-Behnke corneal dystrophy diagnosed by gene-mutation analysis.nnnDESIGNnRetrospective, single-center clinical study.nnnMETHODSnBetween July 2001 and May 2010, 10 consecutive PTKs were performed in 10 eyes of 5 patients (2 male, 3 female; mean age: 55 ± 13 years) with superficially accentuated opacities caused by Thiel-Behnke corneal dystrophy and were followed up for at least 12 months (range: 12-108 months). Main outcome measures included (1) best-corrected visual acuity (BCVA), (2) uncorrected visual acuity (UCVA), (3) spherical equivalent, and (4) recurrence rate. The probability of recurrence of Thiel-Behnke corneal dystrophy after PTK was calculated using the Kaplan-Meier method for survival analysis.nnnRESULTSnThe p.Arg555Gln mutation was found within the TGFBI gene in all 5 patients. Average logarithm of minimal angle of resolution (logMAR) BCVA change was -0.55 ± 0.26. Average logarithm UCVA change was -0.54 ± 0.31. In 5 of the 10 eyes, recurrence of central superficial opacification was clinically identified during the follow-up periods, and in 4 of those 5 eyes, the level of the recurrence was so significant that the visual acuity was reduced more than 2 lines. The maximum follow-up period of the 1 eye without significant post-PTK recurrence was 108 months.nnnCONCLUSIONSnPTK is a successful therapy for Thiel-Behnke corneal dystrophy, and results in midterm stable visual acuity and corneal transparency. Unlike in Reis-Bücklers corneal dystrophy cases, PTK delays the need for more invasive surgical intervention in Thiel-Behnke corneal dystrophy.

PAX6 Isoforms, along with Reprogramming Factors, Differentially Regulate the Induction of Cornea-specific Genes

PAX6 is the key transcription factor involved in eye development in humans, but the differential functions of the two PAX6 isoforms, isoform-a and isoform-b, are largely unknown. To reveal their function in the corneal epithelium, PAX6 isoforms, along with reprogramming factors, were transduced into human non-ocular epithelial cells. Herein, we show that the two PAX6 isoforms differentially and cooperatively regulate the expression of genes specific to the structure and functions of the corneal epithelium, particularly keratin 3 (KRT3) and keratin 12 (KRT12). PAX6 isoform-a induced KRT3 expression by targeting its upstream region. KLF4 enhanced this induction. A combination of PAX6 isoform-b, KLF4, and OCT4 induced KRT12 expression. These new findings will contribute to furthering the understanding of the molecular basis of the corneal epithelium specific phenotype.

Discovery of Molecular Markers to Discriminate Corneal Endothelial Cells in the Human Body

The corneal endothelium is a monolayer of hexagonal corneal endothelial cells (CECs) on the inner surface of the cornea. CECs are critical in maintaining corneal transparency through their barrier and pump functions. CECs in vivo have a limited capacity in proliferation, and loss of a significant number of CECs results in corneal edema called bullous keratopathy which can lead to severe visual loss. Corneal transplantation is the most effective method to treat corneal endothelial dysfunction, where it suffers from donor shortage. Therefore, regeneration of CECs from other cell types attracts increasing interests, and specific markers of CECs are crucial to identify actual CECs. However, the currently used markers are far from satisfactory because of their non-specific expression in other cell types. Here, we explored molecular markers to discriminate CECs from other cell types in the human body by integrating the published RNA-seq data of CECs and the FANTOM5 atlas representing diverse range of cell types based on expression patterns. We identified five genes, CLRN1, MRGPRX3, HTR1D, GRIP1 and ZP4 as novel markers of CECs, and the specificities of these genes were successfully confirmed by independent experiments at both the RNA and protein levels. Notably none of them have been documented in the context of CEC function. These markers could be useful for the purification of actual CECs, and also available for the evaluation of the products derived from other cell types. Our results demonstrate an effective approach to identify molecular markers for CECs and open the door for the regeneration of CECs in vitro.

Establishment of a Human Corneal Epithelial Cell Line Lacking the Functional TACSTD2 Gene as an In Vitro Model for Gelatinous Drop-Like Dystrophy

PURPOSEnGelatinous drop-like corneal dystrophy (GDLD) is characterized by subepithelial amyloid deposition that engenders severe vision loss. The exact mechanism of this disease has yet to be elucidated. No fundamental treatment exists. This study was conducted to establish an immortalized corneal epithelial cell line to be used as a GDLD disease model.nnnMETHODSnA corneal tissue specimen was obtained from a GDLD patient during surgery. Corneal epithelial cells were enzymatically separated from the cornea and were dissociated further into single cells. The epithelial cells were immortalized by the lentiviral transduction of the simian virus 40 (SV40) large T antigen and human telomerase reverse transcriptase (hTERT) genes. For the immortalized cells, proliferative kinetics, gene expressions, and functional analyses were performed.nnnRESULTSnThe immortalized corneal epithelial cells continued to proliferate despite cumulative population doubling that exceeded 100. The cells showed almost no sign of senescence and displayed strong colony-forming activity. The cells exhibited a low epithelial barrier function as well as decreased expression of tight-junction-related proteins claudin 1 and 7. Using the immortalized corneal epithelial cells derived from a GDLD patient, we tested the possibility of gene therapy.nnnCONCLUSIONSnWe established an immortalized corneal epithelial cell line from a GDLD patient. The immortalized cells exhibited cellular phenotypes similar to those of in vivo GDLD. The immortalized cells are thought to be useful for the development of new therapies for treating GDLD corneas and for elucidation of the pathophysiology of GDLD.

The existence of dead cells in donor corneal endothelium preserved with storage media

Aim To investigate the viability of donor corneal endothelial cells (CECs) preserved in storage media by histological examination. Methods Twenty-eight donor corneas were obtained from SightLife Eye Bank (Seattle, Washington), and redundant peripheral portions of those corneas were used for histological examination after removal of the centre corneal graft for transplantation. To assess cell viability in the corneal endothelium, biostaining experiments were performed using propidium iodide, calcein-AM, Hoechst 33u2009342, annexin V, anti-vimentin antibody and toluidine blue. Results Histological analysis of the endothelium showed that the cytoplasm of dead cells had low-intensity fluorescence and that their nuclei stained red, while almost all living cells had green cytoplasm and blue-stained nuclei. The mean dead cell rate in the 28 donor corneas was 4.9%±3.3% (mean ±SD) (range: 0.6%–10.5%). The propidium iodide-positive cells stained positive for annexin V, negative for vimentin and pale for toluidine blue. After the specimens were incubated in a culture medium, the red nucleus dead cells dropped off from the level of the blue nucleus living cells. Conclusion Our findings showed the existence of dead cells in storage-media-preserved donor corneal endothelium and that they dropped off after incubation, thus suggesting that the decrease of CECs following keratoplasty may be related to the presence of dead cells.

Evaluation of corneal neovascularization using optical coherence tomography angiography in patients with limbal stem-cell deficiency.

Purpose: Detection of the exact area of corneal neovascularization using slit-lamp photography is often difficult. Thus, we evaluated corneal neovascularization in patients with limbal stem cell deficiency using optical coherence tomography angiography (OCTA). Methods: Five patients with 5 eyes showing partial or total limbal stem cell deficiency were enrolled. Three eyes had severe corneal scarring. Five 6- × 6-mm images (frontal, upper, lower, nasal, and temporal) were obtained by OCTA. Slit-lamp photography was performed for all patients on the same day. Results: OCTA has 2 advantages over slit-lamp photography for clear demonstration of corneal neovascularization. First, OCTA can show neovascularization in cases with severe corneal opacification. Second, OCTA can detect not only large vessels but also small vessels that cannot be seen by slit-lamp photography. Conclusions: OCTA is a powerful tool for objective evaluation of vascularization in the anterior and posterior segments of the eye. We have demonstrated that OCTA can visualize corneal neovascularization in patients with corneal diseases more clearly than slit-lamp photography.

Restricted Presence of POU6F2 in Human Corneal Endothelial Cells Uncovered by Extension of the Promoter-level Expression Atlas

Corneal endothelial cells (CECs) are essential for maintaining the clarity of the cornea. Because CECs have limited proliferative ability, interest is growing in their potentially therapeutic regeneration from pluripotent stem cells. However, the molecular mechanisms of human CEC differentiation remain largely unknown. To determine the key regulators of CEC characteristics, here we generated a comprehensive promoter-level expression profile of human CECs, using cap analysis of gene expression (CAGE) with a single molecule sequencer. Integration with the FANTOM5 promoter-level expression atlas, which includes transcriptome profiles of various human tissues and cells, enabled us to identify 45 promoters at 28 gene loci that are specifically expressed in CECs. We further discovered that the expression of transcription factor POU class 6 homeobox 2 (POU6F2) is restricted to CECs, and upregulated during human CEC differentiation, suggesting that POU6F2 is pivotal to terminal differentiation of CECs. These CEC-specific promoters would be useful for the assessment of fully differentiated CECs derived from pluripotent stem cells. These findings promote the development of corneal regenerative medicine.