Keratoconus (KC) is a progressive corneal thinning disease that manifests in puberty and worsens during pregnancy. KC onset and progression are attributed to diverse factors that include: environmental, genetics, and hormonal imbalances; however, the pathobiology remains elusive. This study aims to determine the role of corneal stroma sex hormone receptors in KC and their interplay with estrone (E1) and estriol (E3) using our established 3D in vitro model. Healthy cornea stromal cells (HCFs) and KC cornea stromal cells (HKCs), both male and female, were stimulated with various concentrations of E1 and E3. Significant changes were observed between cell types, as well as between males and females in the sex hormone receptors tested; androgen receptor (AR), progesterone receptor (PR), estrogen receptor alpha (ERα), and estrogen receptor beta (ERβ) using Western blot analysis. E1 and E3 stimulations in HCF females showed AR, PR, and ERβ were significantly upregulated compared to HCF males. In contrast, ERα and ERβ had significantly higher expression in HKC’s females than HKC’s males. Our data suggest that the human cornea is a sex-dependent, hormone-responsive tissue that is significantly influenced by E1 and E3. Therefore, it is plausible that E1, E3, and sex hormone receptors are involved in the KC pathobiology, warranting further investigation.

Salivary exosomes have demonstrated vast therapeutic and diagnostic potential in numerous diseases. This study pioneers previously unexplored roles of SE in the context of corneal wound healing by utilizing primary corneal stromal cells from healthy (HCFs), type I diabetes mellitus (T1DMs), type II DM (T2DMs), and keratoconus (HKCs) subjects. Purified, healthy human SEs carrying tetraspanins CD9+, CD63+, and CD81+ were utilized. Scratch and cell migration assays were performed after 0, 6, 12, 24, and 48 h following SE stimulation (5 and 25 µg/mL). Significantly slower wound closure was observed at 6 and 12 h in HCFs with 5 μg/mL SE and T1DMs with 5 and 25 μg/mL SE. All wounds were closed by 24-hour, post-wounding. HKCs, T1DMs, and T2DMs with 25µg/mL SE exhibited a significant upregulation of cleaved vimentin compared to controls. Thrombospondin 1 was significantly upregulated in HCFs, HKCs, and T2DMs with 25 µg/mL SE. Lastly, HKCs, T1DMs, and T2DMs exhibited a significant downregulation of fibronectin with 25 μg/mL SE. Whether SEs can be utilized to clinical settings in restoring corneal defects is unknown. This is the first-ever study exploring the role of SEs in corneal wound healing. While the sample size was small, results are highly novel and provide a strong foundation for future studies.

Biomechanical and molecular stresses may contribute to the pathogenesis of keratoconus (KC). We aimed to profile the transcriptomic changes in healthy primary human corneal (HCF) and KC-derived cells (HKC) combined with TGFβ1 treatment and cyclic mechanical stretch (CMS), mimicking the pathophysiological condition in KC. HCFs (n = 4) and HKCs (n = 4) were cultured in flexible-bottom collagen-coated 6-well plates treated with 0, 5, and 10 ng/mL of TGFβ1 with or without 15% CMS (1 cycle/s, 24 h) using a computer-controlled Flexcell FX-6000T Tension system. We used stranded total RNA-Seq to profile expression changes in 48 HCF/HKC samples (100 bp PE, 70–90 million reads per sample), followed by bioinformatics analysis using an established pipeline with Partek Flow software. A multi-factor ANOVA model, including KC, TGFβ1 treatment, and CMS, was used to identify differentially expressed genes (DEGs, |fold change| ≥ 1.5, FDR ≤ 0.1, CPM ≥ 10 in ≥1 sample) in HKCs (n = 24) vs. HCFs (n = 24) and those responsive to TGFβ1 and/or CMS. PANTHER classification system and the DAVID bioinformatics resources were used to identify significantly enriched pathways (FDR ≤ 0.05). Using multi-factorial ANOVA analyses, 479 DEGs were identified in HKCs vs. HCFs including TGFβ1 treatment and CMS as cofactors. Among these DEGs, 199 KC-altered genes were responsive to TGFβ1, thirteen were responsive to CMS, and six were responsive to TGFβ1 and CMS. Pathway analyses using PANTHER and DAVID indicated the enrichment of genes involved in numerous KC-relevant functions, including but not limited to degradation of extracellular matrix, inflammatory response, apoptotic processes, WNT signaling, collagen fibril organization, and cytoskeletal structure organization. TGFβ1-responsive KC DEGs were also enriched in these. CMS-responsive KC-altered genes such as OBSCN, CLU, HDAC5, AK4, ITGA10, and F2RL1 were identified. Some KC-altered genes, such as CLU and F2RL1, were identified to be responsive to both TGFβ1 and CMS. For the first time, our multi-factorial RNA-Seq study has identified many KC-relevant genes and pathways in HKCs with TGFβ1 treatment under CMS, suggesting a potential role of TGFβ1 and biomechanical stretch in KC development.

Exosomes are a group of vesicles that package and transport DNA, RNA, proteins, and lipids to recipient cells. They can be derived from blood, saliva, urine, and/or other biological tissues. Their impact on several diseases, such as neurodegenerative, autoimmune, and ocular diseases, have been reported, but not fully unraveled. The exosomes that are derived from saliva are less studied, but offer significant advantages over exosomes from other sources, due to their accessibility and ease of collection. Thus, their role in the pathophysiology of diseases is largely unknown. In the context of ocular diseases, salivary exosomes have been under-utilized, thus creating an enormous gap in the literature. The current review discusses the state of exosomes research on systemic and ocular diseases and highlights the role and potential of salivary exosomes as future ocular therapeutic vehicles.

The purpose of this study was to investigate the role of sphingosine kinase 1 (SphK1), which generates sphingosine-1-phosphate (S1P), in corneal neovascularization (NV). Wild-type (WT) and Sphk1 knockout (Sphk1^−/−) mice received corneal alkali-burn treatment to induce corneal NV by placing a 2 mm round piece of Whatman No. 1 filter paper soaked in 1N NaOH on the center of the cornea for 20 s. Corneal sphingolipid species were extracted and identified using liquid chromatography/mass spectrometry (LC/MS). The total number of tip cells and those positive for ethynyl deoxy uridine (EdU) were quantified. Immunocytochemistry was done to examine whether pericytes were present on newly forming blood vessels. Cytokine signaling and angiogenic markers were compared between the two groups using multiplex assays. Data were analyzed using appropriate statistical tests. Here, we show that ablation of SphK1 can significantly reduce NV invasion in the cornea following injury. Corneal sphingolipid analysis showed that total levels of ceramides, monohexosyl ceramides (HexCer), and sphingomyelin were significantly elevated in Sphk^−/− corneas compared to WT corneas, with a comparable level of sphingosine among the two genotypes. The numbers of total and proliferating endothelial tip cells were also lower in the Sphk1^−/− corneas following injury. This study underscores the role of S1P in post-injury corneal NV and raises further questions about the roles played by ceramide, HexCer, and sphingomyelin in regulating corneal NV. Further studies are needed to unravel the role played by bioactive sphingolipids in maintenance of corneal transparency and clear vision.

We have previously reported the flavonoid, quercetin, as a metabolic regulator and inhibitor of myofibroblast differentiation in vitro. Our current study evaluated the effects of topical application of quercetin on corneal scar development using two different animal models followed by RNA analysis in vitro. Wild-type C57BL/6J mice were anesthetized and the corneal epithelium and stroma were manually debrided, followed by quercetin (0.5, 1, 5, or 50 mM) or vehicle application. Corneal scarring was assessed for 3 weeks by slit lamp imaging and clinically scored. In a separate animal study, six New Zealand White rabbits underwent lamellar keratectomy surgery, followed by treatment with 5 mM quercetin or vehicle twice daily for three days. Stromal backscattering was assessed at week 3 by in vivo confocal microscopy. In mice, a single dose of 5 mM quercetin reduced corneal scar formation. In rabbits, stromal backscattering was substantially lower in two out of three animals in the quercetin-treated group. In vitro studies of human corneal fibroblasts showed that quercetin modulated select factors of the transforming growth factor-β (TGF-β) signaling pathway. These results provide evidence that quercetin may inhibit corneal scarring. Further studies in a larger cohort are required to validate the efficacy and safety of quercetin for clinical applications.