Crosstalk of MAP3K1 and EGFR signaling mediates gene-environment interactions that block developmental tissue closure.

Aberrant regulation of signal transduction pathways can adversely derail biological processes for tissue development. One such process is the embryonic eyelid closure that is dependent on the mitogen-activated protein kinase kinase kinase 1 (MAP3K1). Map3k1 KO in mice results in defective eyelid closure and an autosomal recessive eye-open at birth phenotype. We have shown that in utero exposure to dioxin, a persistent environmental toxicant, induces the same eye defect in Map3k1+/- heterozygous but not WT pups. Here, we explore the mechanisms of the Map3k1 (gene) and dioxin (environment) interactions (GxE) underlying defective eyelid closure. We show that, acting through the aryl hydrocarbon receptor, dioxin activates epidermal growth factor receptor signaling, which in turn depresses MAP3K1-dependent Jun N-terminal kinase (JNK) activity. The dioxin-mediated JNK repression is moderate but is exacerbated by Map3k1 heterozygosity. Therefore, dioxin exposed Map3k1+/- embryonic eyelids have a marked reduction of JNK activity, accelerated differentiation and impeded polarization in the epithelial cells. Knocking out Ahr or Egfr in eyelid epithelium attenuates the open-eye defects in dioxin-treated Map3k1+/- pups, whereas knockout of Jnk1 and S1pr that encodes the sphigosin-1-phosphate (S1P) receptors upstream of the MAP3K1-JNK pathway potentiates the dioxin toxicity. Our novel findings show that the crosstalk of aryl hydrocarbon receptor, epidermal growth factor receptor, and S1P-MAP3K1-JNK pathways determines the outcome of dioxin exposure. Thus, gene mutations targeting these pathways are potential risk factors for the toxicity of environmental chemicals.

Epithelial SOX11 regulates eyelid closure during embryonic eye development.

Fibroblast growth factor (FGF10)-mediated signals are essential for embryonic eyelid closure in mammals. Systemic SOX11-deficient mice are born with unclosed eyelids, suggesting a possible role of SOX11 in eyelid closure. However, the underlying mechanisms of this process remain unclear. In this study, we show that epithelial deficiency of SOX11 causes a defect in the extension of the leading edge of the eyelid, leading to failure of embryonic eyelid closure. c-Jun in the eyelid is a transcription factor downstream of FGF10 required for the extension of the leading edge of the eyelid, and c-Jun level was decreased in epithelial SOX11-deficient embryos. These results suggest that epithelial SOX11 plays an important role in embryonic eyelid closure.

Prolonged Distribution of Tranilast in the Eyes after Topical Application onto Eyelid Skin.

Tranilast, a lipophilic drug with various ophthalmic applications, was used as a model drug to establish the possibility of delivering lipophilic drugs through the eyelid skin. Pharmacokinetics and tissue distribution studies were conducted employing three application methods (topical application onto eyelid skin, eye drops, and intravenous injection in rats) to broaden the significance of delivering drugs through the eyelids. A two-compartment open model analysis was used for intravenous route while a non-compartmental evaluation was used for topical applications to estimate the pharmacokinetic parameters. Eyelid skin application, eye drops, and intravenous administration had mean residence times (MRTs) of 8.07, 1.79, and 3.25 h in the eyeball and 10.8, 1.29, and 2.97 h in the conjunctiva, correspondingly. In the eyeball, topical application of tranilast onto the eyelids corresponded to a 4.5- and 2.5-fold higher MRT compared with eye drops and intravenous administration, respectively. An 8.4- or 3.6-fold higher MRT was observed in the conjunctiva after topical application compared with eye drops or intravenous administration, respectively. This indicated a gradual penetration of tranilast into the eyeball and conjunctiva, subsequently a slow elimination from these target tissues.

Clinical, histopathological, and in silico pathogenicity analyses in a pedigree with familial amyloidosis of the Finnish type (Meretoja syndrome) caused by a novel gelsolin mutation.

Purpose: Familial amyloidosis of the Finnish type (FAF) is an inherited amyloidosis arising from mutations in the gelsolin protein (GSN). The disease includes facial paralysis, loose skin, and lattice corneal dystrophy. To date, FAF has been invariably associated with substitution of Asp214 in GSN. We describe the clinical, histopathological, and genetic features of a family with FAF due to a novel GSN mutation. Methods: Five affected adult individuals in a three-generation FAF pedigree were included in the study. Histopathological analysis was performed on an eyelid skin biopsy from one patient. Genetic analysis included next-generation sequencing (NGS) and Sanger sequencing for confirmation of the GSN variant. Several tools for in silico analysis of pathogenicity for the novel variant and to predict the effect of the amino acid replacement on protein stability were used. Results: Three older adult affected patients exhibited corneal lattice dystrophy, cutis laxa, and facultative peripheral neuropathy. Two younger adult individuals presented only with corneal amyloid deposits. NGS identified a heterozygous GSN c.1631T>G transversion, predicting a novel p.Met544Arg mutation. All in silico tools indicated that p.Met544Arg is deleterious for GSN functionality or stability. Conclusions: The results expand the molecular spectrum of GSN-linked systemic amyloidosis. The novel p.Met544Arg pathogenic variant is predicted to affect gelsolin function, presumably by impairing a potential calcium-sensitive, actin-binding region.

Expression of prostaglandin E2 receptor 3 in the eyelid epidermis of patients with Stevens-Johnson syndrome/toxic epidermal necrolysis.

BACKGROUND/AIMS: In a previous genome-wide association study of Stevens-Johnson syndrome/toxic epidermal necrolysis (SJS/TEN) patients we reported the association between SJS/TEN and the prostaglandin E receptor 3 (PTGER3) gene, and that its protein PGE2 receptor 3 (EP3) was markedly downregulated in the conjunctival epithelium of SJS/TEN patients. Here we examined EP3 expression of the eyelid epidermis in SJS/TEN patients with severe ocular complications and investigated the function of EP3. METHODS: For the immunohistochemical study, we obtained eyelid samples from five SJS/TEN patients and five patients without SJS/TEN (control subjects) who were undergoing surgery to treat trichiasis, and investigated the expression of EP3 protein in the epidermis of those samples. To investigate the EP3 function in the human epidermal keratinocytes, we performed ELISA and quantitative reverse transcription polymerase chain reaction, since it is reported that PGE2 suppresses cytokine production via EP3 in human conjunctival epithelium. RESULTS: The results of the immunohistochemical study revealed that EP3 expression in the eyelid epidermis of the SJS/TEN patients was the same as that in the controls. PGE2 and a selective EP3 agonist suppressed cytokine production and expression induced by polyinosine-polycytidylic acid stimulation, such as chemokine ligand 5 and chemokine motif ligand 10. CONCLUSION: Our findings revealed that in chronic-phase SJS/TEN, EP3 protein was expressed in the eyelid epidermis and was not downregulated, unlike in conjunctival epithelium, and that PGE2 could suppress cytokine production via EP3 in human epidermal keratinocytes. Thus, EP3 expression in the epidermis might contribute to a silencing of skin inflammation in chronic-phase SJS/TEN.

Effects of collagen crosslinking on porcine and human tarsal plate.

BACKGROUND: Floppy eyelid syndrome is a disorder in which the tarsal plate is easily distensible and is currently treated with conservative or surgical measures. Human tarsal plate contains type I collagen, which is crosslinked in corneal tissue as a treatment for keratoconus. We hypothesized that collagen crosslinking would similarly stiffen tarsal plate tissue and investigated this in porcine and human tarsal plate specimens. METHODS: Riboflavin-sensitized porcine and human tarsus samples were irradiated with ultraviolet-A light. Porcine experiments were analyzed with gross photographs, anterior segment optical computed tomography (AS-OCT) imaging, and tensile testing. A prospective study of human tarsus was performed on samples from patients undergoing wedge resection for floppy eyelid syndrome and was analyzed with AS-OCT and tensile testing. RESULTS: 73 porcine adnexa and 9 patients (16 eyelids) who underwent wedge excision were included in the study. Grossly, greater stiffness was observed in crosslinked porcine tissue. AS-OCT imaging in porcine tissue showed a distinct hyperreflective band in crosslinked specimens whose area and intensity increased with longer treatment time (P = 0.003); this band was also visible in crosslinked human specimens. Tensile testing was performed, but results were not statistically significant. CONCLUSIONS: AS-OCT imaging, which has not been previously described for tarsal plate, showed a characteristic change in crosslinked porcine and human specimens. Tissue stiffness was increased grossly, but changes in tensile properties were not statistically significant. Further study is warranted to determine relevance as a potential treatment for floppy eyelid syndrome.

Relating evolutionary selection and mutant clonal dynamics in normal epithelia.

Cancer develops from mutated cells in normal tissues. Whether somatic mutations alter normal cell dynamics is key to understanding cancer risk and guiding interventions to reduce it. An analysis of the first incomplete moment of size distributions of clones carrying cancer-associated mutations in normal human eyelid skin gives a good fit with neutral drift, arguing mutations do not affect cell fate. However, this suggestion conflicts with genetic evidence in the same dataset that argues for strong positive selection of a subset of mutations. This implies cells carrying these mutations have a competitive advantage over normal cells, leading to large clonal expansions within the tissue. In the normal epithelium, clone growth is constrained by the limited size of the proliferating compartment and competition with surrounding cells. We show that if these factors are taken into account, the first incomplete moment of the clone size distribution is unable to exclude non-neutral behaviour. Furthermore, experimental factors can make a non-neutral clone size distribution appear neutral. We validate these principles with a new experimental dataset showing that when experiments are appropriately designed, the first incomplete moment can be a useful indicator of non-neutral competition. Finally, we discuss the complex relationship between mutant clone sizes and genetic selection.

Clinical characterization and identification of five novel FOXL2 pathogenic variants in a cohort of 12 Mexican subjects with the syndrome of blepharophimosis-ptosis-epicanthus inversus.

Blepharophimosis-ptosis-epicanthus inversus syndrome (BPES) is an autosomal dominant entity characterized by eyelid malformations and caused by mutations in the forkhead box L2 (FOXL2) gene. Clinical and genetic analyses of large cohorts of BPES patients from different ethnic origins are important for a better characterization of FOXL2 mutational landscape. The purpose of this study is to describe the phenotypic features and the causal FOXL2 variants in a Mexican cohort of BPES patients. A total of 12 individuals with typical facial findings were included. Clinical evaluation included palpebral measurements and levator function assessment. The complete coding sequence of FOXL2 was amplified by PCR and subsequently analyzed by Sanger sequencing. A total of 11 distinct FOXL2 pathogenic variants were identified in our cohort (molecular diagnostic rate of 92%), including 5 novel mutations. Our results broaden the BPES-related mutational spectrum and supports considerable FOXL2 allelic heterogeneity in our population.

Pharmacokinetics and Tissue Distribution of Pilocarpine After Application to Eyelid Skin of Rats.

Extending the delivery of drugs into the eyes while reducing systemic bioavailability is of utmost importance in the management of chronic ocular diseases. Topical application onto the lower eyelid skin, as an alternative to eye drops, is seen to be a valuable strategy in the treatment of chronic eye diseases. To elucidate the critical value of delivering drugs in solution onto the eyeball through the eyelid skin, pharmacokinetic studies of pilocarpine were conducted, and the results were verified using a direct pharmacodynamic study in rats. The mean residence time of pilocarpine after topical eyelid application to the eyelid skin, conjunctiva, eyeball, and plasma were 14.9, 8.50, 6.29, and 8.11 h, respectively. Conjunctiva and eyeball concentrations of pilocarpine at 8 h were 80-fold and 8-fold higher after topical eyelid application, respectively, than those for eye drops. Pupillary constriction was sustained over 8 h after topical eyelid application. Topical eyelid skin application exhibited a localized drug absorption and specific drug accumulation in the ocular tissues. Hence, it is rational to prepare topical formulations directed onto the eyelid skin, which is suitable for drugs required for long-term treatment.

Hyaluronic Acid Gel Biodegradation After Intrapalpebral and Intraorbital Injection in Experimental Study.

PURPOSE: Amid the increasing clinical application of hyaluronic acid (HA) fillers in the ocular adnexa is a paucity of histological data concerning the fate of the injected material. The current study documents the in vivo biodegradation of HA deposited in the eyelid and orbit. METHODS: The study included 22 chinchilla rabbits. The right upper eyelid of 12 rabbits received a single 0.2 ml Restylane (Galderma, Uppsala, Sweden) subcutaneous injection. In 10 different rabbits, the right orbit was injected with 1.0 ml Restylane SubQ (Galderma, Uppsala, Sweden) in the extraconal space. The rabbits in the eyelid group were euthanized at 2 weeks, 1 month, 2, 4, 6, and 9 months, while the rabbits in the orbit group were euthanized at 1 month, 3, 6, 12, and 18 months. Histological analysis was performed on the harvested samples. RESULTS: In the eyelid, the HA assumed a sponge-like structure that diminished gradually over time. At 9 months, the injected HA partially persisted, mainly in the peripheral areas of injection. A similar histologic pattern was observed in the injected orbits, with slow changes persisting at the eighteenth month. In both cohorts, clear signs of collagen deposition and pseudocapsule formation were observed around HA droplets, with no signs inflammation. CONCLUSIONS: HA injected subcutaneously into the eyelid and orbit of rabbits undergoes slow and gradual biodegradation, with HA persisting to no less than 9 months in the eyelid and 18 months in orbit. Neocollagen synthesis and lack of hyaluronidase activity could explain the unexpectedly prolonged HA persistence.

Nuclear Factor Kappa-B Is Enriched in Eyelid Specimens of Rosacea: Implications for Pathogenesis and Therapy.

PURPOSE: To assess the role of nuclear factor kappa-B (NFKB) in cutaneous specimens of rosacea and unaffected tissue. METHODS: Immunohistochemical staining was performed for the activated, phosphorylated variant of NFKB (pNFKB) in eyelid specimens of rosacea (n = 12) and normal, healthy tissue (n = 12). The numbers of positively staining cells/40× microscopic field were counted across 5 consecutive fields. Additionally, quantitative Western blotting was carried out for pNFKB and NFKB in specimens of rosacea (n = 15) and normal controls (n = 14). Statistical comparisons were performed via a dedicated software package. RESULTS: The mean number of cells/40× microscopic field that stained positively for pNFKB was 18.4 (standard deviation = 15.3) for control patients and 39.3 (standard deviation = 16.9) for rosacea patients, and the difference between the 2 groups was statistically significant (P = .0024). On Western blotting, the mean ratios of pNFKB:NFKB for control and rosacea patients measured 0.58 (standard deviation = 0.81) and 3.11 (standard deviation = 3.53), respectively. The 2 groups were statistically significantly different (P = .0002). CONCLUSIONS: The activated form of NFKB is enriched in rosacea, indicating a role for this pathway in the pathogenesis of this disease. Interference with NFKB signaling may represent a novel therapy for rosacea as clinical agents become available. NOTE: Publication of this article is sponsored by the American Ophthalmological Society.

Lipidomic analysis of meibomian gland secretions from the tree shrew: Identification of candidate tear lipids critical for reducing evaporation.

Lipids secreted from the meibomian glands form the outermost layer of the tear film and reduce its evaporation. Abnormal changes in the quantities or compositions of lipids present in meibomian gland secretions (meibum) are known to lead to dry eye disease, although the underlying mechanism is not yet well understood. The tree shrew is the non-primate mammal most closely related to humans. To assess the utility of the tree shrew as a model for the study of dry eye disease, we analyzed the lipid profile of tree shrew meibum using an untargeted ESI-MS and MS/MSall shotgun approach. The resulting lipidome shared many similarities with human meibum, while displaying some interesting differences. For example, several classes of lipids, including wax esters, cholesteryl esters, diesters, and (O-acyl)-ω-hydroxy fatty acids, had relatively longer chain lengths in tree shrew meibum. These increases in length may promote more effective reduction of tear evaporation in the tree shrew, which likely underlies the much longer blinking interval of this mammal. Our results suggest that the tree shrew could be an effective model for the study of dry eye.

Exploring the Asian ethnic predisposition to dry eye disease in a pediatric population.

PURPOSE: To investigate the differences in ocular surface characteristics, tear film parameters, and dry eye symptomology between co-located pediatric populations of Asian and Caucasian ethnicity. METHODS: Seventy New Zealand-born pediatric participants, aged between 5 and 18 years, were recruited in an age and environmentally controlled cross-sectional study. Participants were classified into three groups according to ethnicity and eyelid morphology: Asian single lid (ASL), Asian double lid (ADL), and Caucasian double lid (CDL). Ocular biometry, tear film parameters, ocular surface characteristics, and dry eye symptomology were evaluated in a single clinical session. RESULTS: Overall, no significant intergroup differences were observed in tear film quality, dry eye symptomology, and meibomian gland dropout. A higher proportion of ASL and ADL participants exhibited incomplete blinking than the Caucasian group (both p < 0.001). Meibomian gland shortening was more frequently observed among the two Asian groups (both p < 0.05), while gland tortuosity was more common in the Caucasian group (both p < 0.001). ASL participants exhibited greater inferior lid wiper epitheliopathy grades than ADL participants (p = 0.01), and corneal astigmatism was more pronounced in the ASL than CDL group (p = 0.02). CONCLUSIONS: Ethnic differences in meibomian gland morphological patterns were observed in the current pediatric cohort, although overall meibomian gland dropout did not differ between groups. Asian participants exhibited a higher degree of incomplete blinking, and more marked inferior lid wiper epitheliopathy and corneal astigmatism were observed in the ASL group. These findings would suggest that eyelid anatomy and tension may potentially be implicated in the development of ethnic differences in dry eye disease later in life.

Comparative transcriptome profiles of Lindian chicken eyelids identify melanin genes controlling eyelid pigmentation.

1. During the long history of chicken domestication, eyelid colour, like skin colour and shank colour, has been one of the physical traits of Chinese indigenous chickens that influence consumer buying behaviour. In China, the Lindian chicken, which has coloured feathers, is renowned for the appetizing flavour of its meat and eggs, and its eyelid colours vary from deep (black) to light shades (light yellow). 2. To investigate genes involved in eyelid colour, the expression profiles of black and light-yellow eyelids of Lindian chickens were analysed with transcriptome sequencing. 3. A total of 13 466 genes were detected in the eyelids, among which 14 were differentially expressed. Among these differentially expressed genes (DEGs), three key genes, premelanosome protein (PMEL), dopachrome tautomerase (DCT), and tyrosinase (TYR), encoded proteins that positively regulate melanogenesis and melanin deposition. PMEL, DCT and TYR were expressed much more strongly in the black eyelids than in the light-yellow eyelids. A Kyoto Encyclopedia of Genes and Genomes pathway analysis showed that tyrosine metabolism and melanogenesis genes were significantly enriched among these DEGs (corrected P < 0.05). 4. In conclusion, melanin may be one of the main factors involved in Lindian chicken eyelid colour. Furthermore, these results provide a valuable resource for the future study of the physical traits of Lindian chicken.

Lid wiper epitheliopathy: The influence of multiple lid eversions and exposure time.

PURPOSE: To investigate the effect of multiple lid eversions on lid wiper epitheliopathy (LWE), along with the effect of cumulative lid exposure time and the patterns of associated staining. METHODS: The increase in area of lid wiper staining with lissamine green was compared by everting both the upper eyelids of each subject (i.e. contralateral design), with one eye being everted once for 45 s and the fellow eyelid everted three times, each time for 15 s. This pattern of contralateral eversion was repeated with a total of three eversions in one eye and nine eversions in the fellow eye, with each eye totalling 135 s cumulative exposure to eversion over about 9 min. The LWE area of staining was objectively quantified from slit lamp photography images captured at every lid eversion by 2 masked observers. Two-way repeated measures ANOVAs were used to determine the effect of number of lid eversions and cumulative exposure time on the amount of staining caused. Each image was also categorized into its primary LWE staining pattern, by a masked observer. RESULTS: The multiple eversions condition caused significantly greater LWE than the single eversion condition (p < 0.001), while cumulative exposure time did not have a significant effect on LWE (p = 0.137). Classification of the primary staining patterns revealed that with more eyelid eversions there was a shift from mostly 'no staining' to minor patterns ('short horizontal bands' and 'vertical streaks') and then to more extensive patterns ('broad horizontal bands' and 'comb-shaped'). CONCLUSIONS: The number of eyelid eversions is a confounding factor that should be controlled when investigating LWE, in particular when considering the link with dry eye or contact lens discomfort. However the cumulative exposure time did not appear to influence the LWE magnitude.

Hypoxia: A breath of fresh air for the meibomian gland.

PURPOSE: Optimal meibomian gland (MG) function is critically important for the health and wellbeing of the ocular surface. We hypothesize that low oxygen (O2) conditions promote the function of human MG epithelial cells (HMGECs) and that human MGs exist in a relatively hypoxic environment. The purpose of this study was to test our hypotheses. METHODS: We used human and mouse eyelid segments, and immortalized human MG epithelial cells (IHMGECs) in our studies. To evaluate oxygen (O2) levels in the mouse MG and vicinity, we injected pimonidazole (pimo), a hypoxia marker, before sacrifice. Human eyelid samples were stained with the hypoxia marker glucose transporter 1 (Glut-1). To determine the effect of low O2 levels on IHMGECs, we cultured cells under proliferating and differentiating conditions in both normoxic (21% O2) and hypoxic (3% O2) conditions for 5-15 days. IHMGECs were evaluated for cell number, neutral lipid content, lysosome accumulation, expression of biomarker proteins and DNase II activity. RESULTS: Our results demonstrate that human and mouse MGs, but not the surrounding connective tissue, exist in a relatively hypoxic environment in vivo. In addition, our findings show that hypoxia does not influence IHMGEC numbers in basal or proliferating culture conditions, but does stimulate the expression of SREBP-1 in differentiating IHMGECs. Hypoxia also significantly increased DNase II activity, and apparently IHMGEC terminal differentiation. CONCLUSIONS: Our Results support our hypotheses, and indicate that relative hypoxia promotes MG function.

Predictive Potential of Eyelids and Tear Film in Determining Symptoms in Contact Lens Wearers.

SIGNIFICANCE: Early diagnosis of clinical markers of contact lens discomfort can help clinicians set realistic expectations and monitor and provide prophylactic management for contact lens wearers. PURPOSE: The purpose of this study was to evaluate the potential of eyelid- and tear film-related clinical markers to be used as predictive factors for diagnosing discomfort in contact lens wearers. METHODS: A cross-sectional study was performed on 30 contact lens wearers (6 male, 24 female) with median age of 23 years (range, 18 to 41 years). Eyelid signs and tear film characteristics were evaluated during a single visit, and subjects completed the Contact Lens Dry Eye Questionnaire to evaluate ocular discomfort. Area under the curve (AUC) statistics and sensitivity and specificity values from receiver operating characteristic curves were analyzed to evaluate the predictive potential of clinical signs in discriminating symptoms of contact lens discomfort. RESULTS: The presence of foam at meibomian gland orifices (AUC, 0.944; P < .05; sensitivity >0.7), meibomian gland secretion volume (AUC, 0.935; P < .05; sensitivity >0.7), quality (AUC, 0.969; P < .05; sensitivity >0.7), and expressibility (AUC, 0.933; P < .05; sensitivity >0.7) were significant and strong predictors of discomfort in lens wear. Tear evaporation rates with (AUC, 0.779; P < .05; sensitivity >0.7) or without contact lenses (AUC, 0.788; P < .05; sensitivity >0.7), palpebral conjunctival roughness (AUC, 0.859; P < .05; sensitivity >0.7), palpebral conjunctival staining (AUC, 0.817; P < .05; sensitivity >0.7), palpebral conjunctival hyperemia (AUC, 0.746; P < .05; sensitivity >0.7), meibomian gland orifice capping (AUC, 0.873; P < .05; sensitivity >0.7), pouting (AUC, 0.891; P < .05; sensitivity >0.7), and lid-parallel conjunctival folds (AUC, 0.770; P < .05; sensitivity >0.7) were other acceptable discriminators of symptoms of discomfort during contact lens wear. An equation was developed to identify symptomatic from asymptomatic lens wearers based on the significant predictors: Symptom discriminant function score = 3.378 (meibomian gland secretion grade) + 0.224 (meibomian gland morphology grade) + 0.61 (tear evaporation rate without contact lenses) + 0.439 (lid-parallel conjunctival folds grade) - 0.346 (palpebral conjunctival health grade) - 4.625. CONCLUSIONS: This study demonstrated that clinical signs related to meibomian gland secretions and morphology, tear evaporation, lid-parallel conjunctival folds, and palpebral conjunctival health may successfully predict symptoms of discomfort in contact lens wearers.

Plasma gelsolin promotes re-epithelialization.

Woundhealing disorders characterized by impaired or delayed re-epithelialization are a serious medical problem that is painful and difficult to treat. Gelsolin (GSN), a known actin modulator, supports epithelial cell regeneration and apoptosis. The aim of this study was to estimate the potential of recombinant gelsolin (rhu-pGSN) for ocular surface regeneration to establish a novel therapy for delayed or complicated wound healing. We analyzed the influence of gelsolin on cell proliferation and wound healing in vitro, in vivo/ex vivo and by gene knockdown. Gelsolin is expressed in all tested tissues of the ocular system as shown by molecular analysis. The concentration of GSN is significantly increased in tear fluid samples of patients with dry eye disease. rhu-pGSN induces cell proliferation and faster wound healing in vitro as well as in vivo/ex vivo. TGF-ß dependent transcription of SMA is significantly decreased after GSN gene knockdown. Gelsolin is an inherent protein of the ocular system and is secreted into the tear fluid. Our results show a positive effect on corneal cell proliferation and wound healing. Furthermore, GSN regulates the synthesis of SMA in myofibroblasts, which establishes GSN as a key protein of TGF-ß dependent cell differentiation.

SFTA3 - a novel surfactant protein of the ocular surface and its role in corneal wound healing and tear film surface tension.

The study aimed to characterize the expression and function of SFTA3 at the ocular surface and in tears. Ocular tissues, conjunctival (HCjE) and human corneal (HCE) epithelial cell lines as well as tearfilm of patients suffering from different forms of dry eye disease (DED) were analyzed by means of RT-PCR, western blot, immunohistochemistry, and ELISA. A possible role of recombinant SFTA3 in corneal wound healing was investigated performing in vitro scratch assays. Tear film regulatory properties were analyzed with the spinning drop method and the regulation of SFTA3 transcripts was studied in HCE and HCjE after incubation with proinflammatory cytokines as well as typical ocular pathogens by real-time RT-PCR and ELISA. The results reveal that human ocular tissue as well as tears of healthy volunteers express SFTA3 whereas tears from patients with DED showed significantly increased SFTA3 levels. In vitro wounding of HCE cell cultures that had been treated with recombinant SFTA3 demonstrated a significantly increased wound closure rate and rSFTA3 reduced the surface tension of tear fluid. The results indicate that SFTA3 at the ocular surface seemed to be involved in wound healing and the reduction of surface tension.