Self-complementary AAV vector therapy for treating corneal cloudiness of mucopolysaccharidosis type VII (MPS VII).

PURPOSE: To design a novel efficacious scAAV-Gusb viral vector for treating Mucopolysaccharidosis Type VII (MPS VII) caused by a mutation in the ß-Glu gene (Gusb allele). METHODS: ß-Glu expression of single-stranded AAV-Gusb (ssAAV-Gusb) and self-complementary AAV (scAAV-Gusb) vectors are tested with cultured murine Gusb fibroblasts. The scAAV-Gusb vector was chosen in further studies to prolong the life span and treat corneal pathology of Gusb mice via intrahepatic injection of neonates and intrastromal injection in adults, respectively. Corneal pathology was studied using HRT2 in vivo confocal microscope and histochemistry in mice corneas. RESULTS: Both ssAAV-Gusb and scAAV-Gusb vectors expressed murine ß-Glu in cultured Gusb fibroblasts. The scAAV-Gusb vector had higher transduction efficiency than the ssAAV-Gusb vector. To prolong the life span of Gusb mice, neonates (3 days old) were administered with scAAV-Gusb virus via intrahepatic injection. The treatment improves the survival rate of Gusb mice, prolonging the median survival rate from 22.5 weeks (untreated) to 50 weeks (treated). Thereafter, we determined the efficacy of the scAAV-Gusb virus in ameliorating corneal cloudiness observed in aged Gusb mice. Both corneal cloudiness and stroma thickness decreased, and there was the presence of ß-Glu enzyme activity in the Gusb corneas receiving scAAV-Gusb virus associated with morphology change of amoeboid stromal cells in untreated to characteristic dendritic keratocytes morphology after 4-12 weeks of scAAV-Gusb virus injection. CONCLUSION: Intrahepatic injection of scAAV-Gusb is efficacious in prolonging the life span of Gusb mice, and intrastromal injection can ameliorate corneal phenotypes. Both strategies can be adapted for treating other MPS.

The Effects of ROCK Inhibitor on Prevention of Dexamethasone-Induced Glaucoma Phenotype in Human Trabecular Meshwork Cells.

Purpose: This study investigated the effects of dexamethasone (Dex) on human trabecular meshwork (TM) cells, a model of glucocorticoid-induced glaucoma, and evaluated the impact of ripasudil (Rip) as a co-delivery or sequential dosing strategy. Methods: In vitro experiments were conducted to assess the effects of Dex and Rip on TM cells. Confocal microscopy was used to evaluate the impact of Dex and Rip on F-actin staining signals. Contractility of the TM cells upon Dex and Rip treatment mimicking co-delivery and sequential delivery was quantified using collagen gel contraction assay. Transepithelial electrical resistance (TEER) values and fluorescein isothiocyanate (FITC)-dextran permeability were also measured to assess the impact of Dex and Rip on TM cells. Results: Dex and Rip did not exhibit cytotoxicity at the maximum tested concentration (20 µM). Dex-treated TM cells exhibited higher F-actin staining signals compared to controls, which were reduced when co-treated with Rip. Rip inhibited Dex-induced collagen gel contraction activity in both co-delivery and sequential treatments. Dex resulted in increased TEER values as the dose increased, whereas TEER values were maintained when co-treated with Rip. Conclusions: Co-delivery of Rip has the potential to prevent glaucoma symptoms when patients are treated with Dex. This study highlights the importance of identifying strategies to reduce the side effects of prolonged use of glucocorticoids, such as Dex, in the treatment of various diseases. Translational Relevance: This study demonstrates the potential of co-delivering ripasudil with dexamethasone to mitigate glucocorticoid-induced ocular hypertension and a secondary glaucoma that resembles primary open-angle glaucoma, providing insights for the development of novel preventive strategies in clinical care.

Conditional deletion of CD25 in the corneal epithelium reveals sex differences in barrier disruption.

PURPOSE: IL-2 promotes activation, clonal expansion, and deletion of T cells. IL-2 signals through its heterotrimeric receptor (IL-2R) consisting of the CD25, CD122 and CD132 chains. CD25 knockout (KO) mice develop Sjögren Syndrome-like disease. This study investigates whether corneal CD25/IL-2 signaling is critical for ocular health. METHODS: Eyes from C57BL/6 mice were collected and prepared for immunostaining or in-situ hybridization. Bulk RNA sequencing was performed on the corneal epithelium from wild-type and CD25KO mice. We generated a conditional corneal-specific deletion of CD25 in the corneal epithelium (CD25Δ/ΔCEpi). Corneal barrier function was evaluated based on the uptake of a fluorescent dye. Mice were subjected to unilateral corneal debridement, followed by epithelial closure over time. RESULTS: In C57BL/6 mice, CD25 mRNA was expressed in ocular tissues. Protein expression of CD25, CD122, and CD132 was confirmed in the corneal epithelium. Delayed corneal re-epithelization was seen in female but not male CD25KO mice. There were 771 differentially expressed genes in the corneal epithelium of CD25KO compared to wild-type mice. While barrier function is disrupted in CD25Δ/ΔCEpi mice, re-epithelialization rates are not delayed. CONCLUSIONS: All three chains of the IL-2R are expressed in the corneal epithelium. Our results indicate for the first time, deleting CD25 systemically in all tissues in the mouse and deleting CD25 locally in just the corneal epithelium compromises corneal epithelial barrier function, leading to dry eye disease in female mice. Future studies are needed to delineate the pathways used by IL-2 signaling to influence cornea homeostasis.

Manufacturing of human corneal endothelial grafts.

PURPOSE: Human corneal endothelial cells (HCECs) play a significant role in maintaining visual function. However, these cells are notorious for their limited proliferative capacity in vivo. Current treatment of corneal endothelial dysfunction resorts to corneal transplantation. Herein we describe an ex vivo engineering method to manufacture HCEC grafts suitable for transplantation through reprogramming into neural crest progenitors. METHODS: HCECs were isolated by collagenase A from stripped Descemet membrane of cadaveric corneoscleral rims, and induced reprogramming via knockdown with p120 and Kaiso siRNAs on collagen IV-coated atelocollagen. Engineered HCEC grafts were released after assessing their identity, potency, viability, purity and sterility. Phase contrast was used for monitoring cell shape, graft size, and cell density. Immunostaining was used to determine the normal HCEC phenotype with expression of N-cadherin, ZO-1, ATPase, acetyl-α-tubulin, γ-tubulin, p75NTR, α-catenin, ß-catenin, and F-actin. Stability of manufactured HCEC graft was evaluated after transit and storage for up to 3 weeks. The pump function of HCEC grafts was measured by lactate efflux. RESULTS: One HCEC graft suitable for corneal transplantation was generated from 1/8th of the donor corneoscleral rim with normal hexagonal cell shape, density, and phenotype. The manufactured grafts were stable for up to 3 weeks at 37 °C or up to 1 week at 22 °C in MESCM medium and after transcontinental shipping at room temperature by retaining normal morphology (hexagonal, >2000 cells/mm2, >8 mm diameter), phenotype, and pump function. CONCLUSIONS: This regenerative strategy through knockdown with p120 and Kaiso siRNAs can be used to manufacture HCEC grafts with normal phenotype, morphology and pump function following prolonged storage and shipping.

The versatile roles of lumican in eye diseases: A review.

Lumican is a keratan sulfate proteoglycan that belongs to the small leucine-rich proteoglycan family. Research has lifted the veil on the versatile roles of lumican in the pathogenesis of eye diseases. Lumican has pivotal roles in the maintenance of physiological tissue homogenesis and is often upregulated in pathological conditions, e.g., fibrosis, scar tissue formation in injured tissues, persistent inflammatory responses and immune anomaly, etc. Herein, we will review literature regarding the role of lumican in pathogenesis of inherited congenital and acquired eye diseases, e.g., cornea dystrophy, cataract, glaucoma and chorioretinal diseases, etc.

The gene therapy for corneal pathology with novel nonsense cystinosis mouse lines created by CRISPR Gene Editing.

PURPOSE: Cystinosis is an autosomal recessive lysosomal storage disease (LSDs) caused by mutations in the gene encoding cystinosin (CTNS) that leads to cystine crystal accumulation in the lysosome that compromises cellular functions resulting in tissue damage and organ failure, especially in kidneys and eyes. However, the underlying molecular mechanism of its pathogenesis remains elusive. Two novel mice lines created via CRISPR are used to examine the pathogenesis of cystinosis in the kidney and cornea and the treatment efficacy of corneal pathology using self-complimentary Adeno-associated viral (scAAV-CTNS) vector. METHODS: The CRISPR technique generated two novel cystinotic mouse lines, Ctnsis1 (an insertional mutation) and Ctnsis2 (a nonsense mutation). Immune histochemistry, renal functions test and HRT2 in vivo confocal microscopy were used to evaluate the age-related renal pathogenesis and treatment efficacy of the scAAV-CTNS virus in corneal pathology. RESULTS: Both mutations lead to the production of truncated Ctns proteins. Ctnsis1 and Ctnsis 2 mice exhibit the characteristic of cystinotic corneal crystal phenotype at four-week-old. Treatment with the scAAV-CTNS viral vector decreased the corneal crystals in the treated mice cornea. Ctnsis 1 show renal abnormalities manifested by increased urine volume, reduced urine osmolality, and the loss of response to Desmopressin (dDAVP) at 22-month-old but Ctnsis2 don't manifest renal pathology up to 2 years of age. CONCLUSIONS: Both Ctnsis1 and Ctnsis2 mice exhibit phenotypes resembling human intermediate nephropathic and ocular cystinosis, respectively. scAAV-CTNS viral vectors reduce the corneal cystine crystals and have a great potential as a therapeutic strategy for treating patients suffering from cystinosis.

Trends in using mesenchymal stromal/stem cells (MSCs) in treating corneal diseases.

Since their inception in the 1960s-70s, mesenchymal stem/stromal cells (MSCs) have gained interest because of their differentiation potential, anti-inflammatory effects, and immune-modulating properties. Both cell-based and cell-free MSC treatments show healing capacity in injured tissues. Cell-based treatment comprises MSCs and all secreted products, whereas cell-free treatments include only the secreted products. MSCs are therapeutically administered to many damaged organs owing to their efficacy. Specifically, the eye is a unique organ system to study the effects of MSCs, as treatment is easily applied and measured owing to its external location. The eye holds an immune-privileged status, wherein inflammation and immune responses are innately down-regulated. As excessive inflammation in the cornea often leads to fibrosis and irreversible corneal hazing, many studies have investigated the anti-inflammatory and immune-modulating capacities of MSCs. Decades of research suggest that MSCs modulate the immune response by secreting cytokines, growth factors, and extracellular matrix proteins that inhibit the infiltration of inflammatory cells following injury and promote a healing phenotype via M2 macrophage polarization. MSCs have also shown trans-differentiation potential into cornea-specific cell types during the wound healing process, such as corneal epithelial, stromal, or endothelial cells. This review discusses recent investigations of MSC treatment in the cornea, focusing on therapeutic efficacy, mechanisms, and future directions.

Anomaly of cornea and ocular adnexa in spinster homolog 2 (Spns2) knockout mice.

Spinster 2 (Spns2) is a transporter that pumps sphingosine-1-phosphate (S1P), a bioactive lipid mediator synthesized in the cytoplasm, out of cells into the inter cellular space. S1P is a signal that modulates cellular behavior during embryonic development, inflammation and tissue repair, etc. A Spns2-null (KO) mouse is born with failure of eyelid closure (eyelid-open-at birth; EOB) and develop corneal fibrosis in adulthood. It remains elusive whether corneal lesion is caused by exposure to keratitis (lagophthalmos) of EOB phenotype or the loss of Spns2 directly perturbs the corneal tissue morphogenesis and intra-eyelid structures. Therefore, we investigated differences between the cornea and ocular adnexa morphogenesis in KO and wild-type (WT) embryos and adults as well. The loss of Spns2 perturbs cornea morphogenesis during embryonic development as early as E16.5 besides EOB phenotype. Histology showed that the corneal stroma was thinner with less extracellular matrix accumulation, e.g., collagen and keratocan in the KO mouse. Epithelial stratification, expression of keratin 12 and formation of desmosomes and hemidesmosomes were also perturbed in these KO corneas. Lacking Spns2 impaired morphogenesis of the Meibomian glands and of orbicularis oculi muscles. KO glands were labeled for ELOVL4 and PPARγ and were Oil-Red O-positive, suggesting KO acinar cells possessed functionality as the glands. This is the first report on the roles of Spns2 in corneal and Meibomian gland morphogenesis. Corneal tissue destruction in an adult KO mouse might be due to not only lagophthalmos but also to an impaired morphogenesis of cornea, Meibomian glands, and orbicularis oculi muscle.

Expression and role of lumican in acute aortic dissection: A human and mouse study.

INTRODUCTION: Aortic dissection (AD) is a life-threatening emergency, and lumican (LUM) is a potential Biomarker for AD diagnosis. We investigated LUM expression patterns in patients with AD and explored the molecular functions of Lum in AD mice model. METHODS: LUM expression patterns were analyzed using aortic tissues of AD patients, and serum soluble LUM (s-LUM) levels were compared between patients with acute AD (AAD) and chronic AD (CAD). Lum-knockout (Lum-/-) mice were challenged with ß-aminopropionitrile (BAPN) and angiotensin II (Ang II) to induce AD. The survival rate, AD incidence, and aortic aneurysm (AA) in these mice were compared with those in BAPN-Ang II-challenged wildtype (WT) mice. Tgf-ß/Smad2, Mmps, Lum, and Nox expression patterns were examined. RESULTS: LUM expression was detected in the intima and media of the ascending aorta in patients with AAD. Serum s-LUM levels were significantly higher in patients with AAD than CAD. Furthermore, AD-associated mortality and thoracic aortic rupture incidence were significantly higher in the Lum-/- AD mice than in the WT AD mice. However, no significant pathologic changes in AA were observed in the Lum-/- AD mice compared with the WT AD mice. The BAPN-Ang II-challenged WT and Lum-/- AD mice had higher Tgf-ß, p-Smad2, Mmp2, Mmp9, and Nox4 levels than those of non-AD mice. We also found that Lum expression was significantly higher in the BAPN-Ang II-challenged WT in comparison to the unchallenged WT mice. CONCLUSION: LUM expression was altered in patients with AD display increased s-LUM in blood, and Lum-/- mice exhibited augmented AD pathogenesis. These findings support the notion that LUM is a biomarker signifying the pathogenesis of injured aorta seen in AAD. The presence of LUM is essential for maintenance of connective tissue integrity. Future studies should elucidate the mechanisms underlying LUM association in aortic changes.

Lumican deficiency promotes pulmonary arterial remodeling.

Pulmonary arterial hypertension (PAH) is caused by progressive extracellular matrix disorganization and increased pulmonary vascular cell proliferation. Lumican is a member of the small leucine-rich proteoglycan family that controls cell proliferation, and is a potential endogenous modulator of TGF-ß signaling pathway. We show that the decreased lumican protein levels in pulmonary arterial smooth muscle cells (PASMCs) is related to the vascular remodeling and stiffening observed in PAH. The role of lumican in PASMC accumulation and activation in response to pulmonary vascular remodeling remains unclear and we hypothesized that the loss of lumican in PASMCs promotes the development of PAH. Our aim was to establish that lumican plays a pivotal role in modulating pathological vascular remodeling in humans using a rat model of monocrotaline-induced PAH and chronically hypoxic mice. We found that mice with a homozygous deletion of lumican (Lum-/-) showed severe pulmonary arterial remodeling and right ventricular hypertrophy in response to hypoxia, and these effects in mice with chronic hypoxia-induced pulmonary hypertension were successfully treated by the administration of a lumican C-terminal peptide (LumC13C-A, lumikine). We identified a mechanistic link by which lumican signaling prevents the activation of phosphorylated AKT, resulting in the suppression of PASMC proliferation. Lumican deficiency promotes pulmonary arterial remodeling. Administration of lumikine reverses the PAH pathogenesis caused by hypoxia-induced experimental PAH. Lumican is an antiproliferative target that functions to suppress pAKT activation during pathogenesis.

Laser-Activated Drug Implant for Controlled Release to the Posterior Segment of the Eye.

To treat chronic posterior eye diseases, frequent intravitreal injections or sustained-release drug implants are the current standard of care. Sustained-release drug implants often involve burst release of the drugs and the dosage from the implants cannot be controlled after implantation, which may lead to local side effects. The present study attempts to develop a dosage-controllable drug delivery implant that consists of a nanoporous biodegradable PLGA capsule and light-activated liposomes. Controllable drug release from the implant was achieved using a pulsed near-infrared (NIR) laser both in vitro and in vivo. The in vitro drug release kinetics from two different initial dose implants, 1000 and 500 µg, was analyzed by fitting zero-order and first-order kinetics, as well as the Korsmeyer-Peppas and Higuchi models. The 1000 and 500 µg implants fit the first-order and zero-order kinetics model, respectively, the best. The multiple drug releases in the vitreous were determined by an in vivo fluorimeter, which was consistent with the in vitro data. The dose released was also clinically relevant. Histology and optical and ultrasound imaging data showed no abnormality in the eyes received implant treatment, suggesting that the drug delivery system was safe to the retina. This on-demand dose-controllable drug delivery system could be potentially used for long-term posterior eye disease treatment to avoid frequent invasive injections.

Keratin expression by corneal and limbal stem cells during development.

Keratins are the forming units of intermediate filaments (IF) that provide mechanical support, and formation of desmosomes between cells and hemi desmosomes with basement membranes for epithelium integrity. Keratin IF are polymers of obligate heterodimer consisting one type I keratin and one type II keratin molecules. There are 54 functional keratin genes in human genome, which are classified into three major groups, i.e., epithelial keratins, hair follicle cell-specific epithelial keratins and hair keratins. Their expression is cell type-specific and developmentally regulated. Corneal epithelium expresses a subgroup of keratins similar to those of epidermal epithelium. Limbal basal stem cells express K5/K14, and K8/K18 and K8/K19 IF suggesting that there probably are two populations of limbal stem cells (LSCs). In human, LSCs at limbal basal layer can directly stratify and differentiate to limbal suprabasal cells that express K3/K12 IF, or centripetally migrate then differentiate to corneal basal transient amplifying cells (TAC) that co-express both K3/K12 and K5/K14 prior to moving upward and assuming suprabasal cells phenotype of only K3/K12 expression that signifies corneal type epithelium differentiation. In rodent, the differentiated cornea epithelial cells express K5/K12 in lieu of K3/K12, because K3 allele exists as a pseudogene and does not encode a functional K3 protein. The basal corneal cells of new-born mice originate from surface ectoderm during embryonic development slowly commit to differentiation of becoming TAC co-expressing K5/K12 and K5/K14 IF. However, the centripetal migration may still occur at a slower rate in young mice, which is accelerated during wound healing. In this review, we will discuss and compare the cornea-specific keratins expression patterns between corneal and epidermal epithelial cells during mouse development, and between human and mouse during development and homeostasis in adult, and pathology caused by a mutation of keratins.

Excess Transforming Growth Factor-α Changed the Cell Properties of Corneal Epithelium and Stroma.

Purpose: This study is to investigate the corneal anomaly caused by excess transforming growth factor-α (TGF-α) during mouse development. Methods: Bitransgenic KeraRT/TGF-α mice, generated via cross-mating tetO-TGF-α and KeraRT mice, were induced to overexpress TGF-α by doxycycline commencing at embryonic day 0 or postnatal day 0 to different developmental stages. Bitransgenic mice with doxycycline induction were defined as TGF-αECK mice (TGF-α excess expression by corneal keratocytes). Mouse eyes were examined by hematoxylin and eosin staining, immunofluorescent staining and transmission electron microscopy. Protein and RNA from mouse cornea were subjected to western blotting and real-time quantitative polymerase chain reaction. Results: In TGF-αECK mice, TGF-α overexpression resulted in corneal opacity. Excess TGF-α initially caused corneal epithelial hyperplasia and subsequent epithelium degeneration as the mouse developed, which was accompanied by gradually diminished K12 expression from the periphery of corneal epithelium and increased K13 expression toward the corneal center. Interestingly, K14 was detected in all layers of corneal epithelium of TGF-αECK mice, whereas it was limited at basal layer of controls. Transmission electron microscopy showed desmosome loss between corneal epithelial cells of TGF-αECK mice. In TGF-αECK mice, keratocan expression was abolished; α-SMA expression was increased while expression of Col1a1, Col1a2, and Col5a1 was diminished. Cell proliferation increased in the corneal epithelium and stroma, but not in the endothelium of TGF-αECK mice. Conclusions: Excess TGF-α had detrimental effects on corneal morphogenesis during mouse development in that it changed the cell fate of corneal epithelial cells to assume conjunctival phenotypic expression of K13, and keratocytes to myofibroblast phenotype.

Repressed Wnt Signaling Accelerates the Aging Process in Mouse Eyes.

PURPOSE: Ocular aging is a natural process of functional decline in vision. When the process reaches a point that compromised vision affects normal daily activity, it manifests as age-related ocular diseases, such as age-related macular degeneration, cataracts, glaucoma, and pseudoexfoliation syndrome. We previously reported that repressed Wnt signaling accelerated the maturation of corneal epithelium during tissue development. Here, we explore the hypothesis that repressed Wnt signaling is associated with accelerated aging in mouse eyes. METHODS: Wnt ligand antagonist secreted frizzled-related protein 1 (sFRP1) was expressed in the corneal stroma by a tissue-specific, inducible, bitransgenic system. Tissue structure was analyzed for signs of aging. Signal transduction analysis was performed to determine the cellular response to sFRP1. RESULTS: Mouse eyes with sFRP1 expression showed signs of accelerated aging, resembling those found in pseudoexfoliation (PEX) syndrome, a known age-related disease. Specific findings include granular deposition on the surface of the anterior lens capsule, pigment loss from the anterior surface of the iris, the presence of fibrillary material in the anterior chamber, and changes in cell size (polymegethism) and shape (pleomorphism) of the corneal endothelial cells. In vitro studies demonstrated that sFRP1 did not inhibit Wnt5a function and that cells responded to sFRP1 and Wnt5a in a very similar manner. CONCLUSION: The expression of sFRP1 accelerates the aging process in mouse eyes and future studies are warranted to elucidate the underlying mechanisms.

Transient expression of Wnt5a elicits ocular features of pseudoexfoliation syndrome in mice.

PURPOSE: Pseudoexfoliation (PEX) syndrome is an age-related systemic disease with ocular manifestations. The development of animal models is critical in order to elucidate the cause of the disease and to test potential treatment regimens. The purpose of this study is to report phenotypes found in mouse eyes injected with Adenovirus coding Wnt5a. Some of the phenotypes resemble those found in PEX patients while others are different. METHODS: Recombinant Adenovirus coding Wnt5a or green fluorescent protein (GFP) were injected into mouse eyes. Two months after the injection, eyes were examined for PEX phenotypes using slit lamp, fluorescence stereomicroscope, histological staining, immunostaining and transmission electron microscope. RESULT: Certain ocular features of PEX syndrome were found in mouse eyes injected with recombinant Adenovirus coding Wnt5a. These features include accumulation of exfoliation-like extracellular material on surfaces of anterior segment structures and its dispersion in the anterior chamber, saw-tooth appearance and disrupted basement membrane of the posterior iris pigment epithelium, iris stromal atrophy and disorganized ciliary zonules. Ultrastructure analysis of the exfoliation material revealed that the microfibril structure found in this model was different from those of PEX patients. CONCLUSION: These features, resembling signs of ocular PEX syndrome in patients, suggest that new information obtained from this study will be helpful for developing better mouse models for PEX syndrome.

Impact of Hyaluronic Acid-Containing Artificial Tear Products on Reepithelialization in an In Vivo Corneal Wound Model.

PURPOSE: To evaluate the effect of 6 commercially available hyaluronic acid (HA)-containing topical artificial tear products on corneal reepithelialization following injury, in an in vivo mouse model. METHODS: Ninety-six C57Bl/6 mice (16 per treatment group; male to female ratio, 1:1 per group) were anesthetized. Epithelial debridement was performed on 1 cornea per animal, and the debrided eye was imaged. A 30 µL masked test solution containing 1 of 6 artificial tear products was instilled, immediately on debridement, and subsequently, every 2 h, for a total of 4 administrations. At 24 h post debridement, corneas were stained with fluorescein and imaged to calculate corneal healing rate (number of fluorescein-negative corneas). RESULTS: All 6 artificial tear products used in this study permitted the initial process of corneal wound healing. However, the corneal reepithelialization rate after 24 h was higher with Hydroxypropyl guar (HPG)/HA (53.33%) compared with other HA-containing artificial tear products [HA1 (12.5%), HA2 (26.67%), HA3 (31.25%), HA4 (6.25%), and HA5 (43.75%)]. The average area and percentage area of reepithelialization after 24 h were also higher with HPG/HA compared with other treatment groups. CONCLUSIONS: Percentage of eyes with complete corneal reepithelialization 24 h post debridement was highest with HPG/HA compared with other HA-containing artificial tear products tested. The results of this study provide additional evidence on the potential benefits of HPG/HA in the management of dry eye and its role in the rapid restoration of a healthy ocular epithelium. However, further studies are required to confirm the effects on human corneal wounds.

Generation and Characterization of a Novel Mouse Line, Keratocan-rtTA (KeraRT), for Corneal Stroma and Tendon Research.

Purpose: We created a novel inducible mouse line Keratocan-rtTA (KeraRT) that allows specific genetic modification in corneal keratocytes and tenocytes during development and in adults. Methods: A gene-targeting vector (Kera- IRES2-rtTA3) was constructed and inserted right after the termination codon of the mouse Kera allele via gene targeting techniques. The resulting KeraRT mouse was crossed to tet-O-Hist1H2B-EGFP (TH2B-EGFP) to obtain KeraRT/TH2B-EGFP compound transgenic mice, in which cells expressing Kera are labeled with green fluorescence protein (GFP) by doxycycline (Dox) induction. The expression patterns of GFP and endogenous Kera were examined in KeraRT/TH2B-EGFP. Moreover, KeraRT was bred with tet-O-TGF-α to generate a double transgenic mouse, KeraRT/tet-O-TGF-α, to overexpress TGF-α in corneal keratocytes upon Dox induction. Results: Strong GFP-labeled cells were detected in corneal stroma, limbs, and tail when KeraRT/TH2B-EGFP mice were fed Dox chow. There was no GFP in any single transgenic KeraRT or TH2B-EGFP mouse. Histological analysis showed that GFP in the cornea was limited to stromal keratocytes of KeraRT/TH2B-EGFP, which is consistent with Kera expression. Induction of GFP occurred in 24 hours and reached a plateau by 7 days after Dox induction. GFP could be detected 3-months after induction of KeraRT/TH2B-EGFP. Ectopic expression of TGF-α in corneal keratocytes caused hyperplasia in the corneal epithelium and stroma. Conclusions: The novel Dox inducible KeraRT driver mouse line is a useful genetic tool for gene manipulation and elucidating gene functions in corneal stroma and tendons of limbs and tail during embryonic development, homeostasis and pathogenesis.

Role of EGF receptor signaling on morphogenesis of eyelid and meibomian glands.

The epidermal growth factor receptor (EGFR) signaling has a pivotal role in the regulation of morphogenesis during development and maintenance of homeostasis in adult eyelid and its adnexa. Studies have demonstrated that during eyelid morphogenesis the EGFR signaling pathway is responsible for keratinocyte and mesenchymal cell proliferation and migration at the eyelid tip. For meibomian gland morphogenesis, EGFR signaling activation stimulates meibomian gland epithelial cell proliferation. EGFR signaling pathway functions through multiple downstream signals such as ERK, Rho/ROCK and integrin and is regulated by a variety of upstream signals including Adam17, GPR48 and FGFR signaling. Herein we review the literature that describe the role of EGFR and its related signaling pathways in eyelid and meibomian gland morphogenesis.

Loss of SLC25A46 causes neurodegeneration by affecting mitochondrial dynamics and energy production in mice.

Recently, we identified biallelic mutations of SLC25A46 in patients with multiple neuropathies. Functional studies revealed that SLC25A46 may play an important role in mitochondrial dynamics by mediating mitochondrial fission. However, the cellular basis and pathogenic mechanism of the SLC25A46-related neuropathies are not fully understood. Thus, we generated a Slc25a46 knock-out mouse model. Mice lacking SLC25A46 displayed severe ataxia, mainly caused by degeneration of Purkinje cells. Increased numbers of small, unmyelinated and degenerated optic nerves as well as loss of retinal ganglion cells indicated optic atrophy. Compound muscle action potentials in peripheral nerves showed peripheral neuropathy associated with degeneration and demyelination in axons. Mutant cerebellar neurons have large mitochondria, which exhibit abnormal distribution and transport. Biochemically mutant mice showed impaired electron transport chain activity and accumulated autophagy markers. Our results suggest that loss of SLC25A46 causes degeneration in neurons by affecting mitochondrial dynamics and energy production.

Lumican Peptides: Rational Design Targeting ALK5/TGFBRI.

Lumican, a small leucine rich proteoglycan (SLRP), is a component of extracellular matrix which also functions as a matrikine regulating multiple cell activities. In the cornea, lumican maintains corneal transparency by regulating collagen fibrillogenesis, promoting corneal epithelial wound healing, regulating gene expression and maintaining corneal homeostasis. We have recently shown that a peptide designed from the 13 C-terminal amino acids of lumican (LumC13) binds to ALK5/TGFBR1 (type1 receptor of TGFß) to promote wound healing. Herein we evaluate the mechanism by which this synthetic C-terminal amphiphilic peptide (LumC13), binds to ALK5. These studies clearly reveal that LumC13-ALK5 form a stable complex. In order to determine the minimal amino acids required for the formation of a stable lumican/ALK5 complex derivatives of LumC13 were designed and their binding to ALK5 investigated in silico. These LumC13 derivatives were tested both in vitro and in vivo to evaluate their ability to promote corneal epithelial cell migration and corneal wound healing, respectively. These validations add to the therapeutic value of LumC13 (Lumikine) and aid its clinical relevance of promoting the healing of corneal epithelium debridement. Moreover, our data validates the efficacy of our computational approach to design active peptides based on interactions of receptor and chemokine/ligand.