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In congenital aniridia, not only limbal epithelial cells but also limbal stromal cells may contribute to the development of aniridia associated keratopathy (AAK). Secondary glaucoma affects 50-75% of patients with congenital aniridia, and prostaglandin analogs are commonly used for conservative treatment. This study aimed to explore the effect of travoprost on corneal limbal stromal cells from healthy (LSCs) and congenital aniridia subjects (AN-LSCs), in vitro. Cells were extracted from aniridia (AN-LSCs) (n=7) and healthy donors (LSCs) (n=7). In culture, the cells were treated with travoprost at concentrations ranging from 0.039-40 μg/mL for 20 minutes. Cell viability, proliferation and migration were determined to assess the effect of travoprost on AN-LSCs and LSCs. Analysis of inflammation-, retinoic acid signaling-, and apoptosis-related genes and proteins was performed using qPCR, Western blot, and ELISA. One-way ANOVA was used to analyze cell viability and proliferation. The Mann-Whitney test was applied to compare between-group differences, while the Friedman test was used to assess within-group differences. Both in LSCs and AN-LSCs, travoprost treatment at 0.078 μg/mL and higher concentrations significantly reduced cell viability (p≤0.033; p<0.001) and proliferation decreased both in LSCs and AN-LSCs at 40 μg/mL travoprost concentration (p=0.006; p=0.002). At 6 and 12 hours, 0.313 μg/mL travoprost significantly increased the migration rate of AN-LSCs (p=0.021; p=0.021). AN-LSCs displayed lower PAX6 and JNK (MAPK8) mRNA (p<0.001) but higher MMP-3, MMP-9, ADH7, FABP5 and VEGFA mRNA levels (p≤0.037) than LSCs. PTGFR and JNK mRNA levels, MMP9 and ADH7 protein levels increased significantly in AN-LSCs after 0.313 μg/mL travoprost treatment (p≤0.039), while NF-κB and ADH7 protein levels decreased significantly in LSCs using 0.313 μg/mL travoprost (p=0.039; p<0.001). Travoprost may affect viability, proliferation, and migration of both LSCs and AN-LSCs, with AN-LSCs exhibiting greater sensitivity than LSCs. Additionally, travoprost may regulate MMP-9 expression in AN-LSCs via the JNK signaling pathway. Furthermore, in AN-LSCs, travoprost treatment does not lead to a decrease in NF-κB and ADH7 protein levels.

Progressive aniridia associated keratopathy is worsening visual acuity of congenital aniridia subjects lifelong. Restoration of PAX6 expression in PAX6 haploinsufficient limbal epithelial cells could be one therapeutic option. In a previous study using aniridia-like CRISPR/Cas9 genome-edited corneal epithelial cells, the antipsychotic drugs duloxetine and ritanserin increased PAX6 mRNA and protein expression. Our purpose was to investigate the effect of duloxetine and ritanserin on cultured primary limbal epithelial cells (pLECs) without and with PAX6 knockdown. pLECs were isolated from 11 aniridia patients and corneoscleral rims of 8 healthy human donors and were treated with 5 µM duloxetine or ritanserin for 24 hours. In addition, pLECs were transfected with small interfering RNA (siRNA) (PAX6 knockdown) in the siRNA-based aniridia cell model and were also treated by 5 µM duloxetine or ritanserin for 24 hours. Gene and protein expression were analyzed using qPCR and Western blot. In both primary aniridia limbal epithelial cells and the siRNA-based aniridia cell model, the expression of PAX6 at the transcriptional or translational level did not show significant changes through duloxetine or ritanserin treatment (p > 0.5). The target genes of PAX6 such as KRT3, KRT12, DSG1 , ALDH1A1, ADH7 , FABP5 , ABCG2 also did not change significantly (p ≥ 0.2). Our study shows that primary cultures of limbal epithelial cells from both aniridia patients and healthy donors were unresponsive to drug treatment. Therefore, our data suggest that different aniridia cell models or cell culture conditions exhibit varying responses to duloxetine and ritanserin. The use of in vivo models could further enhance our understanding of duloxetine and ritanserin treatment in aniridia-associated keratopathy.

Haploinsufficiency of the PAX6 gene is the primary pathogenic mechanism underlying classical congenital aniridia. Notably, at least 50% of patients with this condition develop glaucoma. Prostaglandin analogues, such as travoprost, are widely used to lower intraocular pressure in this patient population. At the same time, limbal epithelial cells (LECs) are believed to play a key role in the development and progression of aniridia-associated keratopathy (AAK). Therefore, the aim of this study was to investigate the effects of travoprost on cell viability, proliferation, migration, and the expression of key genes and proteins in both normal and PAX6-knockdown LECs. Primary human LECs were isolated from seven corneal donors. To simulate the haploinsufficient state characteristic of congenital aniridia, a PAX6-knockdown model, using siRNA was employed. LECs were treated with 0.039-40 μg/mL travoprost concentration for 20 minutes. Cell viability was assessed using the XTT assay, while cell proliferation was evaluated by the BrdU assay. Based on XTT results, 0.156 and 0.313 μg/mL travoprost were selected for further measurements in both LECs and PAX6-knockdown LECs. A scratch assay was conducted to measure cell migration. The expression levels of PAX6, FOSL2, MAPKs, inflammatory markers, caspase-3, and MMP9 were analyzed at both the gene and protein levels using qRT-PCR, Western blot, and ELISA. Travoprost significantly reduced LEC viability at 0.156 μg/mL (p = 0.028), while only higher concentrations (20 and 40 μg/mL) inhibited significantly LEC proliferation (p ≤ 0.004). PAX6-knockdown LECs exhibited reduced migration compared to control cells (p ≤ 0.046); however, treatment with 0.313 μg/mL travoprost significantly enhanced their migration (p = 0.047), accompanied by upregulation of JNK1/2 protein (p = 0.039) and MMP9 mRNA and protein levels (p = 0.021, p = 0.027). PAX6 knockdown led to suppression of inflammation-related genes (p ≤ 0.031) and travoprost did not exacerbate inflammatory responses (p ≥ 0.155). Additionally, 0.313 μg/mL travoprost significantly increased caspase-3 protein levels in PAX6-knockdown LECs (p = 0.044). Travoprost, at specific concentrations, can reduce the viability and proliferation of limbal epithelial cells. At 0.313 μg/mL, it significantly upregulates JNK1/2 and MMP9 expression, thereby enhancing the migratory capacity of PAX6-knockdown LECs. These findings may offer valuable insights for the selection of antiglaucomatous medications in patients with congenital aniridia.

Whilst demonstrated extensively in vitro, the control of cell behaviour via modulation of substrate compliance in live tissues has not been accomplished to date. Here we propose that stem cells can be regulated solely through in situ modulation of tissue biomechanics. By first establishing, via high-resolution Brillouin spectro-microscopy, that the outer edge (limbus) of live human corneas has a substantially lower bulk modulus compared to their centre, we then demonstrate that this difference is associated with limbal epithelial stem cell (LESC) residence and YAP-dependent mechanotransduction. This phenotype-through-biomechanics correlation is further explored in vivo using a rabbit alkali burn model. Specifically, we show that treating the burnt surface of the cornea with collagenase effectively restores the tissue’s mechanical properties and its capacity to support LESCs through mechanisms involving YAP suppression. Overall, these findings have extended implications for understanding stem cell niche biomechanics and its impact on tissue regeneration. The link between how the stiffness of the cornea affects stem cells is unclear. Here, the authors use Brillouin spectro-microscopy to show that mechanical properties of the cornea affect epithelial stem cells and after injury, treating the cornea with collagenase suppresses YAP activation, assisting in regeneration.

Dry eye disease (DED) is a multifactorial disease of the ocular surface, characterized by loss of tear film homeostasis and ocular symptoms, in which neurosensory abnormalities have recently been shown to play an etiological role. Although the role of inflammation has been widely studied in DED, the kinetics of immune cells of the ocular surface in this complex disease are hereto unclear. Herein, we utilized intravital multiphoton imaging on transgenic mice to investigate the 3D morphology and kinetics of conventional dendritic cells (cDCs) and the role of ocular surface sensory nerves in regulating them in both the naïve state and experimental DED. Mice with DED had significantly lower tear secretion ( < 0.01), greater corneal fluorescein staining ( < 0.001), and higher cDC density in the ocular surface ( < 0.05), compared to naïve mice. cDCs in DED mice showed morphological alterations in the limbus, exhibiting smaller surface area ( < 0.001) and volume ( < 0.001) compared to naïve mice. Furthermore, corneal cDCs showed greater sphericity in DED mice compared to naïve mice ( < 0.01). In addition, limbal cDCs displayed significantly increased migratory kinetics in DED, including mean track speed, 3D instantaneous velocity, track length, and displacement, compared to naïve mice (all < 0.05). In mice with DED, cDCs showed a higher meandering index in the limbus compared to central cornea ( < 0.05). In DED, cDCs were less frequently found in contact with nerves in the limbus, peripheral, and central cornea ( < 0.05). cDCs in contact with nerves demonstrated a larger surface area ( < 0.001) and volume ( < 0.001), however, they exhibited less sphericity ( < 0.05) as compared to cDCs not in contact with nerves in naïve mice. Importantly, cDCs in contact with nerves during DED had a decreased track length, displacement, mean track speed, and 3D instantaneous velocity compared to those not in contact with nerves (all < 0.05). Taken together, we present evidence of altered cDC kinetics and 3D morphology in DED. Furthermore, apparent neuronal contact significantly alters cDC kinetics and morphological characteristics, suggesting that ocular surface nerves may play a direct role in mediating immune responses in DED.

Limbal stem cells deficiency (LSCD) is an eye disease caused by the loss of stem cells in the corneal limbus as a succession of an injury due physical, biological, or chemical agents. Current therapies of LSCD are focused on the transplantation of donor corneas or tissue equivalents produced from autologous limbal stem cells. Every year there are waiting millions of patients for the cornea transplantation all over the world and the list is growing due to the relatively low number of cornea donors. On the other hand, the transplantation of tissue or cells into the recipient’s body is associated with the higher risk of possible side effects. The possibility of the application of an indirect treatment using the properties of the paracrine activity of stem cells, would be beneficial for the patients with transplant failures. This study was to evaluate the paracrine effect of mesenchymal stem cells derived from adipose tissue (ADSC) on the viability of limbal epithelial stem cells (LESC). The paracrine effect was assessed by treating LESC with conditioned medium collected from ADSC culture. Cell viability, cytotoxicity, apoptosis and proliferation were evaluated using in vitro assays in standard conditions and induced inflammation. After the exposure to the examined conditions, the expression of genes related to pro- and anti- inflammatory factors was evaluated and compared to the secretion of selected cytokines by ELISA test. Moreover, the changes in LESC phenotype were assessed using of phenotype microarrays. Our findings suggest that paracrine activity of ADSC on LESC promotes its proliferation and has a potential role in mitigation of the adverse impact of inflammation induced by lipopolysaccharide.

PurposeThe purpose of this double-masked, parallel randomised controlled trial was to compare the recurrence rate and other outcomes between conjunctival–limbal autograft (CLAu) and mini-simple limbal epithelial transplantation (mini-SLET) after excision of pterygium.MethodsEligibility criteria for participants was the presence of a primary nasal pterygium extending equally to or greater than two millimetres on the cornea on its horizontal axis from the nasal limbus. The participants were allocated into two groups (CLAu and mini-SLET) using simple randomisation with a table of random numbers. Participants and the outcome assessor were masked to the intervention. The study protocol is listed and available on https://clinicaltrials.gov (Identifier: NCT03363282).ResultsA total of 61 eyes were enrolled in the study, 33 underwent CLAu (group 1) and 28 mini-SLET (group 2), all eyes were analysed in each group. At 2, 3, 6 and 12 months the CLAu group exhibited a recurrence of 0%, 6.1%, 8.1% and 8.1%, while the mini-SLET exhibited a recurrence of 0%, 17.9%, 50% and 53.5% (p<0.05). There were no intraoperative or postoperative complications in either of the two groups.ConclusionThe findings of this study suggest that mini-SLET has a higher recurrence rate and provides no advantage over CLAu in the treatment of primary pterygium.

Limbal stem cell deficiency (LSCD) is a disease resulting from the loss or dysfunction of epithelial stem cells, which seriously impairs sight. Autologous limbal stem cell transplantation is effective in unilateral or partial bilateral disease but not applicable in total bilateral disease. An allogeneic source of transplantable cells for use in total bilateral disease can be obtained from culture of donated cadaveric corneal tissue. We performed a controlled multicenter study to examine the feasibility, safety, and efficacy of allogeneic corneal epithelial stem cells in the treatment of bilateral LSCD. Patients were randomized to receive corneal epithelial stem cells cultured on amniotic membrane (AM): investigational medicinal product (IMP) or control AM only. Patients received systemic immunosuppression. Primary endpoints were safety and visual acuity, secondary endpoint was change in composite ocular surface score (OSS). Sixteen patients were treated and 13 patients completed all assessments. Safety was demonstrated and 9/13 patients had improved visual acuity scores at the end of the trial, with no significant differences between IMP and control groups. Patients in the IMP arm demonstrated significant, sustained improvement in OSS, whereas those in the control arm did not. Serum cytokine levels were measured during and after the period of immune suppression and we identified strongly elevated levels of CXCL8 in the serum of patients with aniridia, which persisted throughout the trial. This first randomized control trial of allogeneic corneal epithelial stem cells in severe bilateral LSCD demonstrates the feasibility and safety of this approach. Stem Cells Translational Medicine 2019;8:323–331 Patients with severe ocular surface disorder received transplants of amniotic membrane with (black bars) or without (gray bars) cadaveric‐donor‐derived cultured limbal stem cells. All patients received immune suppression. Only patients who received transplants containing limbal stem cells showed sustained significant improvements (reductions) in combined ocular surface scores (5 factors scored 0–3 where 0 is a normal eye score).

Previous work demonstrated that supraphysiological glucose remodels TGF-β1 and NF-κB signaling in human limbal stromal cells (LSCs) and congenital aniridia-derived LSCs (AN-LSCs). The present study investigated whether the same metabolic stress also alters apoptotic pathways in these cells. Primary LSCs (n = 12) and AN-LSCs (n = 8) were cultured for 48 hours in DMEM containing either normal (17.5 mM) or high (70 mM) glucose. Apoptosis was quantified by Annexin V/propidium iodide (PI) flow cytometry (FC). Expression levels of apoptosis-related genes-including CASP3/7/8/9/10, BCL2, BID, BAX, CDKN1A (p21), CDKN1B (p27), TNFα, XIAP, and BIRC5 (Survivin)-were assessed by qPCR. Protein levels of these markers were analyzed by FC, and TNFα protein concentrations in culture supernatants were measured by ELISA. High glucose significantly reduced the proportion of apoptotic cells in both LSCs (p = 0.0170) and AN-LSCs (p = 0.0181). In both cell types, CASP8 (p = 0.0448; p = 0.0171) and CASP10 (p = 0.0001; p = 0.0007) mRNA levels decreased, while XIAP (p = 0.0375; p = 0.0442) and BIRC5 (p = 0.0196; p = 0.0003) were upregulated. AN-LSCs additionally showed reductions in CASP3 (p = 0.0138) and CDKN1A (p = 0.0331), and exhibited lower BAX levels than LSCs under high glucose (p = 0.0255). Protein analysis corroborated these findings in AN-LSCs: Caspase-3 (p = 0.0154) and Caspase-8 (p = 0.0257) decreased, while Bcl-2 (p = 0.0284) and Survivin (p = 0.0467) levels increased. XIAP protein levels rose in both LSCs (p = 0.0451) and AN-LSCs (p = 0.0134). A 48-hour exposure to 70 mM glucose induces a marked anti-apoptotic shift in human limbal stromal cells, more pronounced in cells derived from congenital aniridia patients. Together with previous evidence on TGF-β1 and NF-κB regulation, these findings suggest that limbal cells may mount an early protective response to metabolic stress, which could be harnessed therapeutically to manage aniridia-associated keratopathy through coordinated survival and stress pathways.

Congenital aniridia is marked by substantial inflammatory changes to the ocular surface. However the exact mechanisms of epithelial-stromal interaction are not fully understood. The purpose of this study was to investigate inflammatory cytokine expression in limbal epithelial cells and fibroblasts following exposure to each other’s conditioned medium (CM). Healthy primary limbal epithelial cells (pLECs) and healthy (LFC) or aniridia primary limbal fibroblasts (AN-LFC) were isolated. A PAX6-deficient limbal epithelial cell line (mut-LSCs) modeled aniridia. pLECs underwent siRNA-mediated PAX6 knockdown (siPAX6 pLECs) with control cells transfected with non-specific siRNA (siCtrl pLECs). siCtrl and siPAX6 pLECs were treated with LFC-CM and AN-LFC-CM for 24 hours, while LFC and AN-LFC were treated with pLECs-CM and mut-LSCs-CM for 48 hours. Gene and protein expression of IL-1β, IL-6, IL-8, TNF-α and VEGF-A were measured using qPCR and ELISA. Except for an increased IL-8 protein expression in siPAX6 pLECs treated with LFC-CM, gene and protein levels of inflammatory biomarkers remained unchanged in siCtrl and siPAX6 pLECs, regardless of treatment with LFC-CM or AN-LFC-CM. In LFCs, pLECs-CM decreased TNF-α mRNA and IL-8 protein, while increasing IL-1β, IL-6, IL-8 mRNA and IL-6 protein. LFCs treated with mut-LSCs-CM showed decreased TNF-α mRNA and increased IL-6 protein. AN-LFCs treated with pLECs-CM showed increased IL-6, IL-8 and VEGF-A mRNA and IL-6 protein. mut-LSCs-CM did not alter AN-LFC expression. Limbal fibroblasts’ secretome minimally inflames limbal epithelial cells, suggesting a supportive niche role. In contrast, pLECs-CM induces a stronger fibroblast response, indicating abnormal interactions in congenital aniridia.