Epithelial barrier dysfunction in ocular allergy.

The epithelial barrier is the first line of defense that forms a protective barrier against pathogens, pollutants, and allergens. Epithelial barrier dysfunction has been recently implicated in the development of allergic diseases such as asthma, atopic dermatitis, food allergy, and rhinitis. However, there is limited knowledge on epithelial barrier dysfunction in ocular allergy (OA). Since the ocular surface is directly exposed to the environment, it is important to understand the role of ocular epithelia and their dysfunction in OA. Impaired epithelial barrier enhances allergen uptake, which lead to activation of immune responses and development of chronic inflammation as seen in allergies. Abnormal expression of tight junction proteins that helps to maintain epithelial integrity has been reported in OA but sufficient data not available in chronic atopic (AKC) and vernal keratoconjunctivitis (VKC), the pathophysiology of which is not just complex, but also the current treatments are not completely effective. This review provides an overview of studies, which indicates the role of barrier dysfunction in OA, and highlights how ocular barrier dysfunction possibly contributes to the disease pathogenesis. The review also explores the potential of ocular epithelial barrier repair strategies as preventive and therapeutic approach.

Epithelial cells removed in advanced surface ablation (ASA) surgery can be used as a source of corneal samples to perform in vitro studies.

Human corneal epithelial cells are needed to study corneal pathophysiology in vitro. Due to the limitations of cell lines, the use of primary cells is highly desirable, but the scarcity of human tissues, along with ethical issues, make it difficult to accomplish all required experiments. In advanced surface ablation (ASA), the central corneal epithelium is removed and discarded. We hypothesized that ASA samples could be used to perform in vitro assays. In this study, 29 samples from patients undergoing ASA were recovered in supplemented DMEM/F12 culture medium, RIPA buffer, or RLT lysis buffer. The first aim was to determine whether cells could be maintained in culture. Although with the explant technique, tissue pieces did not attach to the culture surface, after disaggregation, cells showed high viability (90.0 ± 6.0%), attached to plates, and remained viable for up to 14 days. The second aim was to elucidate if ASA samples could be used to study protein or gene expression. Cytokeratin-3, ZO-1, Ki67, and E-cadherin protein expression were confirmed by immunofluorescence. Total protein (485.8 ± 115.8 µg) was isolated from cells in RIPA buffer, and GAPDH was detected by Western blotting, indicating that samples are adequate for protein studies. RNA (9.0 ± 3.6 µg) was isolated from samples in RLT lysis buffer, and GAPDH gene expression was studied by PCR, confirming that samples were also suitable for gene expression studies. These results suggest that samples obtained from corneal surface ablation procedures may constitute a valuable source of human cells to accomplish in vitro studies.

Characterization and functional performance of a commercial human conjunctival epithelial cell line.

The conjunctiva is a complex tissue that covers the eye beginning at the corneal limbus and extending over the inner surfaces of the eyelids. Due to its important functions in maintaining the health of the ocular surface, adequate in vitro models of conjunctival structure and function are essential to understand its roll in different pathologies. Because there is scarcity of human conjunctival tissue that can be used in research, cell lines are often the only option for initial studies. An immortalized human conjunctival epithelial cell (IM-HConEpiC) line is now commercially available; however, it is not very well characterized. In this study, we have developed a new protocol to culture these cells without the use of collagen-coated culture surfaces, but with a defined cell culture medium. We characterized IM-HConEpiCs cultured under these conditions and corroborated that the cells maintained a conjunctival epithelial phenotype, including acidic and neutral mucins, junctional proteins E-cadherin and zonula occludens 1, and expression of CK8 and CK19, among others. In addition, we analyzed the response to oxidative stress and inflammatory stimuli and found that IM-HConEpiCs respond as expected for conjunctival epithelial tissue. For instance, cells exposed to oxidative stress increased the production of reactive oxygen species, and that increase was blocked in the presence of an antioxidant agent. In addition, after stimulation with TNF-α, IM-HConEpiCs significantly increased the production of IL-1ß, IL-6, IL-8, and IP-10. Therefore, with this study we conclude that IM-HConEpiCs can be a useful tool in functional studies to determine the response of the conjunctiva to pathological conditions and/or to test new therapeutic strategies.

Ocular Delivery of Polyphenols: Meeting the Unmet Needs.

Nature has become one of the main sources of exploration for researchers that search for new potential molecules to be used in therapy. Polyphenols are emerging as a class of compounds that have attracted the attention of pharmaceutical and biomedical scientists. Thanks to their structural peculiarities, polyphenolic compounds are characterized as good scavengers of free radical species. This, among other medicinal effects, permits them to interfere with different molecular pathways that are involved in the inflammatory process. Unfortunately, many compounds of this class possess low solubility in aqueous solvents and low stability. Ocular pathologies are spread worldwide. It is estimated that every individual at least once in their lifetime experiences some kind of eye disorder. Oxidative stress or inflammatory processes are the basic etiological mechanisms of many ocular pathologies. A variety of polyphenolic compounds have been proved to be efficient in suppressing some of the indicators of these pathologies in in vitro and in vivo models. Further application of polyphenolic compounds in ocular therapy lacks an adequate formulation approach. Therefore, more emphasis should be put in advanced delivery strategies that will overcome the limits of the delivery site as well as the ones related to the polyphenols in use. This review analyzes different drug delivery strategies that are employed for the formulation of polyphenolic compounds when used to treat ocular pathologies related to oxidative stress and inflammation.

Reliability and reproducibility of a rodent model of choroidal neovascularization based on the subretinal injection of polyethylene glycol.

Purpose: To evaluate the reliability and reproducibility of a rodent choroidal neovascularization (CNV) model by subretinal injection of polyethylene glycol (PEG). Methods: C57BL/6 mice were injected subretinally with 2 µl PBS (Gibco, Invitrogen, Paisley, UK; n=14) or PEG (1 mg; n=18). Animals were sacrificed at either 0, 5, 14 or 21 days. Eyes were embedded in paraffin wax and serial sections were stained with haematoxylin and eosin or Fontana-Masson or immunostained for cytokeratin 8/18, isolectin B4 (IB4), vascular endothelial growth factor (VEGF) and von Willebrand factor (vWF). Results: Both the PBS and PEG groups had retinal degeneration and retinal pigment epithelium (RPE)/choroid modifications at 5 and 14 days. Pigment clumps and cell vacuolization at the RPE/choroid were identified as melanin-containing RPE cells. In PEG-injected eyes, CK8/18-positive cellular elements were present at the subretinal space, IB4 immunoreactivity was significantly increased and choroidal vessels appeared diffusely thickened. However, neither VEGF nor vWF (angiogenesis/neovascularization markers) were detected in either group. At 21 days, the retina/choroid of PBS-injected animals was normal in appearance, while retina/choroid changes remained in some PEG-injected mice. Conclusions: Subretinal injection of PEG induced retina/choroid degenerative modifications that mimic the initial steps of human CNV. However, ocular changes were heterogeneous among animals from PBS and PEG groups and did not follow a consistent pattern while most PBS-injected animals showed similar degenerative changes. Abnormal growth of new vessels originating from the choroidal vasculature was not observed. Therefore, we consider that this model does not consistently reproduce CNV and that researchers should choose other rodent models of CNV to avoid misinterpreting their results.

Improved in vitro corneal delivery of a thrombospondin-1-derived peptide using a liposomal formulation.

Peptide delivery to and through ocular sites is a growing field of research interest. However, several barriers restrict the permeation and bioavailability of these molecules to target tissues. The main pharmacological barriers of topical administration are the tear film, rapid drainage of the tear film, and poor corneal permeation. If the administered molecule is a peptide, instability and enzymatic degradation can be significant. Novel approaches such as the design and development of nanocarriers to overcome these drawbacks have been investigated with promising results. Therefore, in continuation of our previous study using a liposome-based (LP) formulation as topical drug delivery system, the aim of this work was to efficiently encapsulate a thrombospondin-1-derived peptide, KRFK, in this formulation and to assess peptide permeability through different ocular surface epithelia. LPs were prepared by the solvent evaporation technique and the labeled peptide FITC-KRFK was incorporated in the aqueous core. Different sonication times were used to optimize encapsulation efficiency. The selected formulation was further analyzed in terms of size, pH, osmolarity, and corneal epithelial cytotoxicity. The permeabilities of the LP-encapsulated and free labeled KRFK peptides were assessed with in vitro models of conjunctival and corneal epithelia. Our results provide a proof of concept that the LP formulation efficiently encapsulates the KRFK peptide and improves corneal permeation. Data reported in this study strongly support that this formulation could be a more effective therapeutic approach than free peptide instillation and warrant further analysis using experimental in vivo models.

Topical Application of TGF-ß-Activating Peptide, KRFK, Prevents Inflammatory Manifestations in the TSP-1-Deficient Mouse Model of Chronic Ocular Inflammation.

Chronic inflammation of the ocular surface poses a risk of vision impairment. The understanding of the molecular mechanisms that are involved in the inflammatory response is critical to identify novel molecular targets. Recently, thrombospondin-1 (TSP-1) has emerged as a key player in ocular surface homeostasis that efficiently activates the TGF-ß2 isoform that is predominantly expressed in the ocular mucosa. Here, the potential of the peptide derived from TSP-1 (KRFK), that can activate TGF-ß, is proposed as a potentially applicable therapeutic for chronic ocular surface inflammatory disorders. Our in vitro results confirm that the chosen peptide activates TGF-ß, reducing the expression of co-stimulatory molecules on dendritic cells, driving them towards a tolerogenic phenotype. For the in vivo studies, the TSP-1-/- mouse is used as a pre-clinical model of chronic ocular inflammation. We observe that the topical application of KRFK alters the peripheral balance of effectors by reducing the proportion of pathogenic Th1 and Th17 cells while increasing Treg cell proportion in cervical lymph nodes. In line with these findings, the development of chronic ocular surface inflammation is significantly prevented in KRFK-treated TSP-1-/- mice, as assessed by clinical parameters and inflammatory cytokine expression in conjunctival and lacrimal gland tissues. Together, our results identify the KRFK peptide as a novel therapeutic option to prevent the development of chronic inflammatory manifestations of the ocular surface.

Novel anti-inflammatory liposomal formulation for the pre-ocular tear film: In vitro and ex vivo functionality studies in corneal epithelial cells.

In ocular surface inflammatory diseases, such as dry eye disease, long-term symptom relief requires targeting the inflammation itself rather than treating only the surface-associated dryness with artificial tears. Therefore, we included an anti-inflammatory agent in an unpreserved liposome-based (LP) formulation used as artificial tears. Our aim was to characterize and study its in vitro and ex vivo cell uptake and functionality. Human corneal epithelial (HCE) cells were used to study MPA-LP-induced effects after 60 min of exposure, using blank LP and non-LP MPA formulations as controls. A fluorescent labeled LP formulation was used to determine uptake by HCE cells and localization in ex vivo porcine corneas. The LP formulation complied with the required physicochemical properties and had no cytotoxicity on HCE cells after 60 min of exposure. HCE cells showed LP-associated fluorescence at 24, 48, and 72 h after 60 min of exposure, and the LP-associated fluorescence was uniformly distributed throughout the porcine corneal epithelium immediately after 5 min of exposure. MPA-LP increased protein expression and nuclear translocation of progesterone receptor in comparison with controls as determined by Western blotting and immunofluorescence. Moreover, MPA-LP significantly reduced the cell proliferation rate and IL-6 and IL-8 production 48 h after the exposure period, as determined by the alamarBlue assay and ELISA, respectively. None of these effects were evident in blank LP-exposed cells and non-LP MPA formulation reduced only IL-6 production. Our results suggest that the LP-based formulation, used to replenish the lipids of the tear film, can be loaded with anti-inflammatory agents that can be delivered into the cells and activate specific drug receptors. These agents can reduce inflammatory cytokine production and may be effective in the treatment of inflammatory processes associated with ocular surface diseases.

Conjunctival Goblet Cell Function: Effect of Contact Lens Wear and Cytokines.

This review focuses on conjunctival goblet cells and their essential function in the maintenance of eye health. The main function of goblet cells is to produce and secrete mucins that lubricate the ocular surface. An excess or a defect in those mucins leads to several alterations that makes goblet cells central players in maintaining the proper mucin balance and ensuring the correct function of ocular surface tissues. A typical pathology that occurs with mucous deficiency is dry eye disease, whereas the classical example of mucous hyperproduction is allergic conjunctivitis. In this review, we analyze how goblet cell number and function can be altered in these diseases and in contact lens (CL) wearers. We found that most published studies focused exclusively on the goblet cell number. However, recent advances have demonstrated that, along with mucin secretion, goblet cells are also able to secrete cytokines and respond to them. We describe the effect of different cytokines on goblet cell proliferation and secretion. We conclude that it is important to further explore the effect of CL wear and cytokines on conjunctival goblet cell function.

Thrombospondin-1 induces differential response in human corneal and conjunctival epithelial cells lines under in vitro inflammatory and apoptotic conditions.

Recently, thrombospondin-1 (TSP-1) has been reported to be critical for maintaining a healthy ocular surface. The purpose of the study was to characterize the expression of TSP-1 and of its receptors CD36 and CD47 in corneal and conjunctival epithelial cells and determine the effect of exogenous TSP-1 treatment on these cells, following the induction of inflammation- and apoptosis-related changes. The expression of TSP-1, CD36 and CD47 by corneal and conjunctival cell lines was firstly characterized by ELISA, immunofluorescence analysis, Western blotting and reverse transcription polymerase chain reaction (RT-PCR). Benzalkonium chloride (BAC) exposure for 5 or 15 min was used as pro-inflammatory and pro-apoptotic stimulus for corneal or conjunctival epithelial cells, respectively. To analyze inflammation and apoptosis-related changes, IL-6 and TGF-ß2 secretion determined by ELISA was used as inflammatory markers, while activated caspase-3/7 levels and cell viability, determined by CellEvent™ Caspase-3/7 Green Detection Reagent and XTT cytotoxicity assay, respectively, were used as apoptotic markers. Changes in CD36 and CD47 mRNA expression were quantified by real time RT-PCR. Corneal epithelial cells secreted and expressed higher protein levels of TSP-1 than conjunctival epithelial cells, although TSP-1 mRNA expression levels were similar and had lower CD36 and CD47, both at protein and mRNA levels. Both cell lines responded to exogenous TSP-1 treatment increasing CD36 at protein and mRNA levels. Blocking experiments revealed a predominance of TSP-1/CD47 rather than TSP-1/CD36 interactions to up-regulate CD36 levels in conjunctival epithelial cells, but not in corneal epithelial cells. BAC exposure increased IL-6 secretion and caspase-3/7 levels and decreased cell viability in both, corneal and conjunctival epithelial cells. Moreover, BAC exposure increased latent TGF-ß2 levels in conjunctival epithelial cells. Interestingly, CD36 mRNA expression was down-regulated after BAC exposure in both cell lines. Exogenous TSP-1 treatment reduced TGF-ß2 up-regulated levels by BAC exposure in conjunctival epithelial cells and less pronounced reduced IL-6 in BAC-exposed corneal epithelial cells. The effect on CD36 and CD47 regulation was less pronounced or even opposite depending on the inflammation- and apoptosis-related markers tested. Our results show evidence of the capacity of corneal and conjunctival epithelial cells to respond to TSP-1 via CD36 or CD47. Experimental simulation of inflammation- and apoptosis-related conditions changed the effects differentially elicited by TSP-1 on corneal and conjunctival epithelial cells, suggesting an unexpected and relevant contribution of TSP-1 on ocular surface homeostasis regulation.

IL-6 as a corneal wound healing mediator in an in vitro scratch assay.

Corneal healing process under inflammatory conditions is not fully understood. We aimed at determining the effect of an inflammatory (presence of IL-6) or anti-inflammatory (presence of IL-10) environment and a mixture of both in the expression of IL-6 signaling pathway mediators, and on corneal wound healing in an in vitro scratch assay. For that purpose, human corneal epithelial cells were cultured until confluence. The effect of IL-6 (10 ng/ml), IL-10 (20 ng/ml) or IL-6 + IL-10 exposure on the expression of IL-6R, gp130, and STAT3 was determined by Western blotting and quantitative PCR, at different time points. The monolayer was mechanically wounded using a sterile 10 µl pipette tip. Wound healing rate in the presence or absence of these cytokines was measured immediately after cytokine exposure and after 4, 8, and 24 h. The effect of mitomycin C on wound healing rate, in control and IL-6-stimulated cells, was also evaluated. Detection of proliferative cells was performed with an EdU imaging kit. For the visualization of migrating cells, cold methanol-fixed cells were incubated with an α-actinin antibody. For the statistical analysis a two-factor design of experiment method was applied. Levene test was used to contrast equality of variances. If variances were equal, ANOVA was performed to test the equality of means. If variances were not equal, a Mood's median test was performed. We observed that IL-6 and IL-10 stimulation, and their combination, increased gp130 production at different time points. STAT3 production was increased in IL-6-stimulated cells, at 72 h. An increase in pSTAT3 production was found in IL-6- and IL-10-stimulated cells, that was sustained in time in IL-6 + IL-10 co-stimulated cultures. Scraped areas had an initial width of 570.57 ± 75.82 µm. In IL-6-exposed cells wound healing closure was faster than in control cells or IL-10-exposed cells. After 8 h, wound width in IL-10-exposed cells, was also significantly smaller than that of control cells. Cells exposed to IL-6 + IL-10 had the slowest wound healing rate, similar to control cells. Wounds were closed after 24 h regardless the experimental condition. Mitomycin C exposure increased the wound closure rate in every experimental condition. No significant differences in the percentage of proliferative cells at the edge of the scratch and in distant areas of the monolayer were found. At the edge of the scratch, some actin filaments of non-proliferative cells were directed through the cell-free area, independently of the stimulating condition. In conclusion, the presence of IL-10 and, most importantly, of IL-6, increased the wound healing rate in an in vitro corneal wound healing model. The combination of both cytokines did not have a synergistic action in wound healing. In our model, wound closure was the result of the combination of cell proliferation and cell migration.

CD44 and RHAMM hyaluronan receptors in human ocular surface inflammation.

BACKGROUND: CD44 and RHAMM hyaluronan (HA) receptors have been studied in several systemic diseases such as osteoarthritis and cancer. However, not too much is known about their role in ocular surface disorders. The purpose of this research was to determine if CD44 and RHAMM are implicated in human ocular surface inflammation. METHODS: Upper tarsal conjunctival epithelial samples from patients with active ocular surface inflammation (n = 17) and healthy controls (n = 14) were recovered by brush cytology. Patients were evaluated by an ophthalmologist and classified in different groups according to the etiology (immune atopic diseases or immune non-atopic diseases) and inflammation intensity (mild/moderate or severe). CD44, RHAMM, and p53 mRNAs were measured using real-time PCR. RESULTS: CD44, RHAMM, and p53 mRNAs were detected in all samples. In immune atopic diseases, higher levels of CD44 and RHAMM mRNAs were present, reaching a 300 % increase for RHAMM in severe inflammation (p < 0.001). In contrast, in immune non-atopic diseases, the HA receptors were downregulated. CD44 tended to decrease up to 30 % in severe patients (p = 0.06), and RHAMM decreased 40 % in severe inflammation (p = 0.021). CONCLUSIONS: RHAMM may be implicated in severe ocular surface inflammation affecting the upper tarsal conjunctiva.

Purinergic receptors in ocular inflammation.

Inflammation is a complex process that implies the interaction between cells and molecular mediators, which, when not properly "tuned," can lead to disease. When inflammation affects the eye, it can produce severe disorders affecting the superficial and internal parts of the visual organ. The nucleoside adenosine and nucleotides including adenine mononucleotides like ADP and ATP and dinucleotides such as P(1),P(4)-diadenosine tetraphosphate (Ap4A), and P(1),P(5)-diadenosine pentaphosphate (Ap5A) are present in different ocular locations and therefore they may contribute/modulate inflammatory processes. Adenosine receptors, in particular A2A adenosine receptors, present anti-inflammatory action in acute and chronic retinal inflammation. Regarding the A3 receptor, selective agonists like N(6)-(3-iodobenzyl)-5'-N-methylcarboxamidoadenosine (CF101) have been used for the treatment of inflammatory ophthalmic diseases such as dry eye and uveoretinitis. Sideways, diverse stimuli (sensory stimulation, large intraocular pressure increases) can produce a release of ATP from ocular sensory innervation or after injury to ocular tissues. Then, ATP will activate purinergic P2 receptors present in sensory nerve endings, the iris, the ciliary body, or other tissues surrounding the anterior chamber of the eye to produce uveitis/endophthalmitis. In summary, adenosine and nucleotides can activate receptors in ocular structures susceptible to suffer from inflammatory processes. This involvement suggests the possible use of purinergic agonists and antagonists as therapeutic targets for ocular inflammation.

Involvement of corneal epithelial cells in the Th17 response in an in vitro bacterial inflammation model.

PURPOSE: Staphylococcus aureus (SA) and Pseudomonas aeruginosa (PA) are frequent causes of bacterial keratitis, an inflammatory process that can lead to vision loss. We used a human corneal epithelial (HCE) cell line to study the Th17 inflammatory pathway, including interleukin (IL-) 6, IL-17, and associated receptors, in response to stimulation by SA and PA culture supernatants. METHODS: Cells of the HCE cell line were exposed to either SA or PA supernatants in dilutions of 1:100 or 1:50, or to human recombinant IL-17A (20 ng/ml). Cell culture supernatants were collected at 6, 24, and 72 h, and protein and RNA were isolated. Expression of cytokine (IL-6, IL-17A), receptor (sIL-6R, IL-17RA), and mediator (soluble glycoprotein [sgp] 130, MIP3α) proteins and mRNAs were determined with enzyme-linked immunosorbent assay, immunohistochemistry, western blotting, and real-time, reverse-transcription quantitative PCR. In addition, IL-17RA was localized by transmission electron microscopy after immunogold labeling. RESULTS: Basal secretion of IL-6 and IL-17A by HCE cells occurred in a time-dependent manner. Expression of IL-6 was significantly enhanced by SA stimulation, but not by PA stimulation. IL-6 mRNA expression was higher in the control and SA-stimulated cells at 6 and 24 h, but not at 72 h. In the PA-stimulated cells, mRNA levels were significantly lower than the controls at 6 and 24 h. Expression of sIL-6R was not altered by SA or PA supernatants, but sgp130 expression was greater than controls at 6 h, less than controls at 24 h, and the same as controls at 72 h. HCE cells secreted IL-17A in a time-dependent manner that was not altered by stimulation; however, the IL-17A mRNA levels were lower than those of the controls at 6 h. With immunohistochemistry, IL-17RA was localized in perinuclear vesicles and in the cytosol and membranes of HCE cells. IL-17RA was also present in the epithelial cells from human ocular surface tissues. As quantified with western blotting, expression of IL-17RA was unchanged in HCE cells stimulated by SA or PA supernatants. CONCLUSIONS: HCE cells react to bacterial inflammation by enhancing the secretion of IL-6 and by regulating the proinflammatory response with differential secretion of sgp130. Under normal conditions, HCE cells and ocular surface tissues express IL-17RA. Additionally, HCE cells express IL-17RA after bacterial stimulation. All of these molecules are involved in the Th17 differentiation pathway, suggesting that corneal epithelial cells may act as indirect participants in the Th17 signaling pathway.

Isolation and Characterization of Human Conjunctival Mesenchymal Stromal Cells and Their Extracellular Vesicles.

Purpose: The purpose of this study was to isolate and culture human conjunctival mesenchymal stromal cells (Conj-MSCs) from cadaveric donor tissue, and to obtain and characterize their extracellular vesicles (EVs) and their effect on conjunctival epithelium. Methods: Stromal cells isolated from cadaveric donor conjunctival tissues were cultured and analyzed to determine whether they could be defined as MSCs. Expression of MSC markers was analyzed by flow cytometry. Cells were cultured in adipogenic, osteogenic, and chondrocyte differentiation media, and stained with Oil Red, Von Kossa, and Toluidine Blue, respectively, to determine multipotent capacity. EVs were isolated from cultured Conj-MSCs by differential ultracentrifugation. EV morphology was evaluated by atomic force microscopy, size distribution analyzed by dynamic light scattering, and EVs were individually characterized by nanoflow cytometry. The effect of EVs on oxidative stress and viability was analyzed in in vitro models using the conjunctival epithelial cell line IM-HConEpiC. Results: Cultured stromal cells fulfilled the criteria of MSCs: adherence to plastic; expression of CD90 (99.95 ± 0.03% positive cells), CD105 (99.04 ± 1.43%), CD73 (99.99 ± 0.19%), CD44 (99.93 ± 0.05%), and absence of CD34, CD11b, CD19, CD45 and HLA-DR (0.82 ± 0.91%); and in vitro differentiation into different lineages. Main Conj-MSC EV subpopulations were round, small EVs that expressed CD9, CD63, CD81, and CD147. Conj-MSC EVs significantly decreased the production of reactive oxygen species in IM-HConEpiCs exposed to H2O2 in similar levels than adipose tissue-MSC-derived EVs and ascorbic acid, used as controls. Conclusions: It is possible to isolate human Conj-MSCs from cadaveric tissue, and to use these cells as a source of small EVs with antioxidant activity on conjunctival epithelial cells.

Hyaluronan receptors in the human ocular surface: a descriptive and comparative study of RHAMM and CD44 in tissues, cell lines and freshly collected samples.

The purpose of this study was to demonstrate the presence of the receptor for hyaluronan-mediated motility (RHAMM) in human conjunctival epithelium and in two widely used cell lines from human corneal (HCE) and conjunctival (IOBA-NHC) epithelia. We compared the distribution of RHAMM proteins and mRNAs in human ocular surface tissues (corneal, limbal and conjunctival), HCE and IOBA-NHC cell lines, and corneal and conjunctival epithelia primary samples from healthy donors with the previously identified hyaluronan receptor CD44. We also aimed to determine if soluble CD44 (sCD44) was present in human tears, as it could have a role in the interaction of the tear fluid with hyaluronan. Protein expression was evaluated by Western blots and immunofluorescence microscopy. mRNA expression was evaluated by RT-PCR and Q-PCR. sCD44 was analyzed by ELISA in culture supernatants and in human tears. We describe the expression of RHAMM in human healthy conjunctiva and in HCE and IOBA-NHC cells at both protein and mRNA levels, and the presence of sCD44 in human tears. Furthermore, we detected CD44 and sCD44 expression variations in in vitro inflammatory conditions. This study also focused on the necessary caution with which the conclusions extracted from cell lines should be made, and in the great value of using primary samples as often as possible.

Extracellular Vesicles from Human Adipose-Derived Mesenchymal Stem Cells: A Review of Common Cargos.

In recent years, the interest in adipose tissue mesenchymal cell-derived extracellular vesicles (AT-MSC-EVs) has increasingly grown. Numerous articles support the potential of human AT-MSC-EVs as a new therapeutic option for treatment of diverse diseases in the musculoskeletal and cardiovascular systems, kidney, skin, and immune system, among others. This approach makes use of the molecules transported inside of EVs, which play an important role in cell communication and in transmission of macromolecules. However, to our knowledge, there is no database where essential information about AT-MSC-EVs cargo molecules is gathered for easy reference. The aim of this study is to describe the different molecules reported so far in AT-MSC- EVs, their main molecular functions, and biological processes in which they are involved. Recently, the presence of 591 proteins and 604 microRNAs (miRNAs) has been described in human AT-MSC-EVs. The main molecular function enabled by both proteins and miRNAs present in human AT-MSC-EVs is the binding function. Signal transduction and gene silencing are the biological processes in which a greater number of proteins and miRNAs from human AT-MSC-EVs are involved, respectively. In this review we highlight the therapeutics effects of AT-MSC-EVs related with their participation in relevant biological processes including inflammation, angiogenesis, cell proliferation, apoptosis and migration, among others.

Improved ocular delivery of quercetin and resveratrol: A comparative study between binary and ternary cyclodextrin complexes.

The number of patients affected by Dry Eye Disease (DED) had notably increased worldwide, addressing the need of novel therapeutic approaches. Polyphenols, quercetin (QUE) and resveratrol (RSV) show necessary antioxidant and anti-inflammatory properties to manage DED, but their application as topical eyedrops is restricted by low aqueous solubility and low chemical stability. Cyclodextrins (CD) are widely used to improve physicochemical characteristics of drugs. Consequently, the aim of this study was to make a comparison between binary complexes with quercetin, resveratrol and cyclodextrins and tertiary complexes adding hyaluronic acid (HA). Both complexes were able to enhance solubility and stability of QUE and RSV. AFM imaging and DLS measurements disclose the formation of spherical nanoaggregates within tertiary complexes of both QUE and RSV with mean diameters of 103 and 82 nm. Neither complex demonstrated cytotoxic effect in in vitro studies in corneal (HCE) and conjunctival (IM-ConjEpi) cell lines. In HCE cells, complexes containing QUE or RSV at their highest concentrations were able to scavenge more than 95 % of the ROS that were produced intracellularly (p < 0.005). Similar response was observed with IM-ConjEpi cells. The antioxidant effect was maintained in the complexes with HA. This confirmed their potential as viable topical treatment for DED.

Potential Ophthalmological Application of Extracts Obtained from Tuna Vitreous Humor Using Lactic Acid-Based Deep Eutectic Systems.

A green technique was developed to extract hyaluronic acid (HA) from tuna vitreous humor (TVH) for its potential application in managing dry eye disease. Deep eutectic solvents (DES) were used to extract HA and were synthesized using natural compounds (lactic acid, fructose, and urea). The DES, the soluble fraction of TVH in DES (SF), and the precipitated extracts (PE) were evaluated for their potential use in dry eye disease treatment. In vitro experiments on human corneal epithelial cell lines and the effect on dry eye-associated microorganisms were performed. The influence of the samples on the HCE viability, their intracellular reactive oxygen species (ROS) scavenging capacity, inflammatory response, and antimicrobial properties were studied. According to the results, all samples displayed an antioxidant effect, which was significantly higher for PE in comparison to SF. Most of the tested samples did not induce an inflammatory response in cells, which confirmed the safety in ophthalmic formulations. In addition, the DES and SF proved to be efficient against the studied bacterial strains, while PE did not show an antimicrobial effect. Hence, both DES and SF at defined concentrations could be used as potential compounds in dry eye disease management.

Intracellular trafficking of hyaluronic acid-chitosan oligomer-based nanoparticles in cultured human ocular surface cells.

PURPOSE: Nanoparticles are a promising alternative for ocular drug delivery, and our group has proposed that they are especially suited for ocular mucosal disorders. The goal of the present study was to determine which internalization pathway is used by cornea-derived and conjunctiva-derived cell lines to take up hyaluronic acid (HA)-chitosan oligomer (CSO)-based nanoparticles (HA-CSO NPs). We also determined if plasmids loaded onto the NPs reached the cell nucleus. METHODS: HA-CSO NPs were made of fluoresceinamine labeled HA and CSO by ionotropic gelation and were conjugated with a model plasmid DNA for secreted alkaline phosphatase. Human epithelial cell lines derived from the conjunctiva and the cornea were exposed to HA-CSO NPs for 1 h and the uptake was investigated in living cells by fluorescence microscopy. The influence of temperature and metabolic inhibition, the effect of blocking hyaluronan receptors, and the inhibition of main endocytic pathways were studied by fluorometry. Additionally, the metabolic pathways implicated in the degradation of HA-CSO NPs were evaluated by lysosome identification. RESULTS: There was intracellular localization of plasmid-loaded HACSO NPs in both corneal and conjunctival cells. The intracellular presence of NPs diminished with time. HA-CSO NP uptake was significantly reduced by inhibition of active transport at 4 °C and by sodium azide. Uptake was also inhibited by blocking hyaluronan receptors with anti-CD44 Hermes-1 antibody, by excess HA, and by filipin, an inhibitor of caveolin-dependent endocytosis. HA-CSO NPs had no effect on cell viability. The transfection efficiency of the model plasmid was significantly higher in NP treated cells than in controls. CONCLUSIONS: HA-CSO NPs were internalized by two different ocular surface cell lines by an active transport mechanism. The uptake was mediated by hyaluronan receptors through a caveolin-dependent endocytic pathway, yielding remarkable transfection efficiency. Most of HA-CSO NPs were metabolized within 48 h. This uptake did not compromise cell viability. These findings further support the potential use of HA-CSO NPs to deliver genetic material to the ocular surface.