[Corrigendum] Melanogenesis in uveal melanoma cells: Effect of argan oil.

Following the publication of the above article, an interested reader drew to the authors' attention that, in Fig. 7 on p. 1282, a pair of the western blotting bands in the Akt blot positioned adjacent to each other looked strikingly similar. Although the authors considered that the data were correct as shown (and the Editorial Office were in agreement that it was not certain that the bands were identical), to avoid any possible confusion or suspicion, the authors requested that the figure be reprinted showing the Akt data obtained from one of the repeated experiments. The revised version of Fig. 7, containing the replacement data for the Akt western blotting data, is shown opposite. All the authors agree with the publication of this corrigendum, and are grateful to the Editor of International Journal of Molecular Medicine for allowing them the opportunity to publish this for the purposes of clarifying the presented data. [International Journal of Molecular Medicine 40: 1277­1284, 2017; DOI: 10.3892/ijmm.2017.3104].

Effects of Topical Gabapentin on Ocular Pain and Tear Secretion.

Neuropathic ocular pain is a frequent occurrence in medium to severe dry eye disease (DED). Only palliative treatments, such as lubricants and anti-inflammatory drugs, are available to alleviate patients' discomfort. Anesthetic drugs are not indicated, because they may interfere with the neural feedback between the cornea and the lacrimal gland, impairing tear production and lacrimation. Gabapentin (GBT) is a structural analog of gamma-amino butyric acid that has been used by systemic administration to provide pain relief in glaucomatous patients. We have already shown in a rabbit model system that its topic administration as eye drops has anti-inflammatory properties. We now present data on rabbits' eyes showing that indeed GBT given topically as eye drops has analgesic but not anesthetic effects. Therefore, opposite to an anesthetic drug such as oxybuprocaine, GBT does not decrease lacrimation, but-unexpectedly-even stimulates it, apparently through the upregulation of acetylcholine and norepinephrine, and by induction of aquaporin 5 (AQP5) expression in the lacrimal gland. Moreover, data obtained in vitro on a primary human corneal epithelial cell line also show direct induction of AQP5 by GBT. This suggests that corneal cells might also contribute to the lacrimal stimulation promoted by GBT and participate with lacrimal glands in the restoration of the tear film, thus reducing friction on the ocular surface, which is a known trigger of ocular pain. In conclusion, GBT is endowed with analgesic, anti-inflammatory and secretagogue properties, all useful to treat neuropathic pain of the ocular surface, especially in case of DED.

Poly 2-methacryloyloxyethyl Phosphorylcholine Protects Corneal Cells and Contact Lenses from Desiccation Damage.

SIGNIFICANCE: Contact lens (CL) wearing may cause discomfort and eye dryness. We describe here the efficacy of a synthetic polymer in protecting both the corneal epithelial cells and the CL from desiccation damage. Artificial tears containing this polymer might be helpful to treat or prevent ocular surface damage in CL wearers. PURPOSE: We aimed to investigate the protective effects of the synthetic polymer 2-methacryloyloxyethyl phosphorylcholine (poly-MPC) on corneal epithelial cells and CLs subjected to desiccation damage. METHODS: The interaction of poly-MPC with the cell membrane was evaluated on human primary corneal epithelial cells (HCE-F) by the sodium dodecyl sulfate damage protection assay or the displacement of the cell-binding lectin concanavalin A (ConA). Survival in vitro of HCE-F cells and ex vivo of porcine corneas exposed to desiccating conditions after pre-treatment with poly-MPC or hyaluronic acid (HA), hypromellose (HPMC), and trehalose was evaluated by a colorimetric assay. Soft CLs were soaked overnight in a solution of poly-MPC/HPMC and then let dry in ambient air. Contact lens weight, morphology, and transparency were periodically registered until complete dryness. RESULTS: Polymer 2-methacryloyloxyethyl phosphorylcholine and HPMC were retained on the HCE-F cell membrane more than trehalose or HA. Polymer 2-methacryloyloxyethyl phosphorylcholine, HA, and HPMC either alone or in association protected corneal cells from desiccation significantly better than did trehalose alone or in association with HA. Contact lens permeation by poly-MPC/HPMC preserved better their shape and transparency than did saline. CONCLUSIONS: Polymer 2-methacryloyloxyethyl phosphorylcholine coats and protects corneal epithelial cells and CLs from desiccation damage more efficiently compared with trehalose and as good as other reference compounds.

The Anti-Inflammatory Effect of the ß1-Adrenergic Receptor Antagonist Metoprolol on High Glucose Treated Human Microvascular Retinal Endothelial Cells.

Hyperglycemia-induced impairment of the blood-retinal barrier represents the main pathological event in diabetic retinopathy that is elicited by a reduced cellular response to an accumulation of reactive oxygen species (ROS) and increased inflammation. The purpose of the study was to evaluate whether the selective ß1-adrenoreceptor (ß1-AR) antagonist metoprolol could modulate the inflammatory response to hyperglycemic conditions. For this purpose, human retinal endothelial cells (HREC) were treated with normal (5 mM) or high glucose (25 mM, HG) in the presence of metoprolol (10 µM), epinephrine (1 µM), or both compounds. Metoprolol prevented both the HG-induced reduction of cell viability (MTT assays) and the modulation of the angiogenic potential of HREC (tube formation assays) reducing the TNF-α, IL-1ß, and VEGF mRNA levels (qRT-PCR). Moreover, metoprolol prevented the increase in phospho-ERK1/2, phospho-cPLA2, COX2, and protein levels (Western blot) as well as counteracting the translocation of ERK1/2 and cPLA2 (high-content screening). Metoprolol reduced ROS accumulation in HG-stimulated HREC by activating the anti-oxidative cellular response mediated by the Keap1/Nrf2/HO-1 pathway. In conclusion, metoprolol exerted a dual effect on HG-stimulated HREC, decreasing the activation of the pro-inflammatory ERK1/2/cPLA2/COX2 axis, and counteracting ROS accumulation by activating the Keap1/Nrf2/HO-1 pathway.

Isolation and Characterization of a New Human Corneal Epithelial Cell Line: HCE-F.

PURPOSE: To isolate and characterize an epithelial cell (EC) line from a human donor cornea, which may serve as a reliable test cell line to address biomolecular issues and study the response of corneal epithelium to stressing events and therapeutic treatments. METHODS: A corneal button from a donor patient was treated with enzymes to separate the epithelial sheet and to free EC, which were put in tissue culture. ECs were characterized by optic and electronic microscopies, cytokeratins and PAX6 were detected by SDS-PAGE and western immunoblotting, the barrier function was evaluated by transepithelial electric resistance and by the immune detection of membrane junction proteins, and the karyotype was characterized according to the classical methods. RESULTS: Morphological analyses returned the picture of classical homogeneous polygonal morphology as expecetd by EC that was maintained over time and several in vitro passages. Transepithelial electric resistance values were characteristic of a typical barrier-forming cell line. The cytokeratin expression pattern was the one expected for corneal EC with a predominance of CK3 and CK5 and different from a human keratocyte cell line. The male karyotype showed 2 trisomies, of chromosomes 8 and 11. CONCLUSIONS: All the data so far obtained with the HCE-F cells concur to certify this cell line as a stable, true primary human corneal EC line, which could then be used as a test cell line to study and address the questions concerning the biological response of human corneal epithelium to stressing and/or therapeutic treatments and as a term of comparison for EC derived from pathological corneas.

Atropine Differentially Modulates ECM Production by Ocular Fibroblasts, and Its Ocular Surface Toxicity Is Blunted by Colostrum.

 Background: The etiology and the mechanism behind atropine treatment of progressive myopia are still poorly understood. Our study addressed the role of scleral and choroidal fibroblasts in myopia development and atropine function. METHODS: Fibroblasts treated in vitro with atropine or 7-methylxanthine were tested for ECM production by Western blotting. Corneal epithelial cells were treated with atropine in the presence or absence of colostrum or fucosyl-lactose, and cell survival was evaluated by the MTT metabolic test. RESULTS: Atropine and 7-methyl-xanthine stimulated collagen I and fibronectin production in scleral fibroblasts, while they inhibited their production in choroidal fibroblasts. Four days of treatment with atropine of corneal epithelial cells significantly decreased cell viability, which could be prevented by the presence of colostrum or fucosyl-lactose. CONCLUSIONS: Our results show that atropine may function in different ways in different eye districts, strengthening the scleral ECM and increasing permeability in the choroid. The finding that colostrum or fucosyl-lactose attenuate the corneal epithelial toxicity after long-term atropine treatment suggests the possibility that both compounds can efficiently blunt its toxicity in children subjected to chronic atropine treatment.

Novel indole derivatives targeting HuR-mRNA complex to counteract high glucose damage in retinal endothelial cells.

The ELAVL1 (or human antigen R - HuR) RNA binding protein stabilizes the mRNA, with an AU-rich element, of several genes such as growth factors (i.e. VEGF) and inflammatory cytokines (i.e. TNFα). We hereby carried out a virtual screening campaign in order to identify and test novel HuR-mRNA disruptors. Best-scored compounds were tested in an in-vitro model of diabetic retinopathy, namely human retinal endothelial cells (HRECs) challenged with high-glucose levels (25 mM). HuR, VEGF and TNFα protein contents were evaluated by western-blot analysis in total cell lysates. VEGF and TNFα released from HRECs were measured in cell medium by ELISA. We found that two derivatives bearing indole moiety, VP12/14 and VP12/110, modulated HuR expression and decreased VEGF and TNF-α release by HREC exposed to high glucose (HG) levels. VP12/14 and VP12/110 inhibited VEGF and TNF-α release in HRECs challenged with high glucose levels, similarly to dihydrotanshinone (DHTS), a small molecule known to interfere with HuR- TNFα mRNA binding. The present findings demonstrated that VP12/14 and VP12/110 are innovative molecules with anti-inflammatory and anti-angiogenic properties, suggesting their potential use as novel candidates for treatment of diabetic retinopathy.

Anti-angiogenic effect of quercetin and its 8-methyl pentamethyl ether derivative in human microvascular endothelial cells.

Angiogenesis is involved in many pathological states such as progression of tumours, retinopathy of prematurity and diabetic retinopathy. The latter is a more complex diabetic complication in which neurodegeneration plays a significant role and a leading cause of blindness. The vascular endothelial growth factor (VEGF) is a powerful pro-angiogenic factor that acts through three tyrosine kinase receptors (VEGFR-1, VEGFR-2 and VEGFR-3). In this work we studied the anti-angiogenic effect of quercetin (Q) and some of its derivates in human microvascular endothelial cells, as a blood retinal barrier model, after stimulation with VEGF-A. We found that a permethylated form of Q, namely 8MQPM, more than the simple Q, is a potent inhibitor of angiogenesis both in vitro and ex vivo. Our results showed that these compounds inhibited cell viability and migration and disrupted the formation of microvessels in rabbit aortic ring. The addition of Q and more significantly 8MQPM caused recoveries or completely re-establish the transendothelial electrical resistance (TEER) to the control values and suppressed the activation of VEGFR2 downstream signalling molecules such as AKT, extracellular signal-regulated kinase, and c-Jun N-terminal kinase. Taken together, these data suggest that 8MQPM might have an important role in the contrast of angiogenesis-related diseases.

Age-Related Dry Eye Lactoferrin and Lactobionic Acid.

Dry eye is the most prominent pathology among those involving the ocular surface: a decrease of the aqueous (less frequent) or the lipid (more frequent) component of the tear film is the cause of the diminished stability of tears that is observed in this pathology. Dry eye shows a clear distribution linked to both sex (being more frequent among women) and age (increasing with aging). Therefore, specific treatments taking into account the etiology of the disease would be desired. The role of lactoferrin and its functional mimetic lactobionic acid are reported here as a possible remedy for age-related dry eye.

Experimental Evidence of the Healing Properties of Lactobionic Acid for Ocular Surface Disease.

PURPOSE: The aim of this study was to investigate the properties of lactobionic acid (LA) as a possible supplement in artificial tears in in vitro and in vivo experimental model systems. LA is a bionic derivative of a polyhydroxy acid, which consists of one galactose attached by an ether link to a gluconic acid. It is a molecule endowed with several properties that make it an ideal supplement in artificial tears: it is highly hygroscopic and a powerful antioxidant, it is an iron chelator and inhibits matrix metalloprotease activity; it favors wound healing (WH); and it inhibits bacterial growth. METHODS: Promotion of WH by LA, alone or in combination with hyaluronic acid (HA), was investigated in vitro on monolayers of rabbit corneal cells (Statens Seruminstitut) and in vivo after epithelium debridement of rabbit corneas. TGF-ß expression and MMP-9 activity in wounded corneas were detected in tears and cornea extracts by western blot or by Enzyme Linked ImmunoSorbent Assay (ELISA). Bacterial growth inhibition by LA was checked on Staphylococcus aureus isolates in liquid culture. RESULTS: LA, with or without HA, favors WH in vitro and in vivo. The WH assay on the rabbit cornea showed that 4% LA in association with 0.15% HA also resulted in a blunted increase of MMP-9 and TGF-ß in tears and corneal tissue. Finally, the presence of 4% LA resulted in slower growth of cultured bacterial isolates. CONCLUSIONS: Our findings support the hypothesis that LA could be a useful supplement to artificial tears to treat ocular surface dysfunction such as dry eye.

Phenotypic characterization of the SIRC (Statens Seruminstitut Rabbit Cornea) cell line reveals a mixed epithelial and fibroblastic nature.

The aim of the present study was to investigate, in the Statens Seruminstitut Rabbit Cornea (SIRC) cell line, the presence of epithelial and fibroblastic markers, comparing their levels with those of the human Retinal Pigmented Epithelial (ARPE-19) cell line, and the Human Keratocyte (HK) cell line, respectively. SIRC cells, often described as of epithelial origin, are used as a corneal epithelial barrier model to study the permeability of ophthalmic drugs. However, they show a morphology that is more consistent with a fibroblastic cell phenotype, similar to corneal keratocytes. Our comparative analyses of cell type specific markers demonstrated that SIRC do not express cytokeratins 19 and 16 (typical of ARPE-19) and cytokeratin 9 (typical of HK); they do express cytokeratins 3 and 18 common to all three cell lines, and cytokeratin 12 typical of ARPE-19. Tight junction proteins were absent in HK, and lower in SIRC than in ARPE-19. All cell lines expressed the markers lumican and vimentin, with SIRC expressing intermediate levels between HK and ARPE-19; alpha-SMA was highly expressed in all lines. These markers, considered typical of fibroblasts, can be, however, expressed by epithelial cells during wound healing. These results might suggest that long-term in vitro cultivation of cell lines leads to a derangement of their specific phenotype, most likely due to genetic and epigenetic factors. This could be the reason why SIRC cells came to exhibit a hybrid nature between epithelial and fibroblastic cells.

N-hydroxymethylglycinate with EDTA is an efficient eye drop preservative with very low toxicity: an in vitro comparative study.

PURPOSE: Preservatives are used in multi-dose ophthalmic topical medications in order to prevent contamination by bacteria and fungi. However, prolonged use of preserved eye drops, as it may happen in dry eye or glaucoma, may damage cells of the ocular surface. Therefore, an important goal is to find preservatives with low toxicity which are mild to host cells, still able to prevent drug contamination so to maintain their sterility and efficacy. Hence, aim of this study has been to compare the relative toxicity on a rabbit corneal cell line of a new preservative, made by the association of N-hydroxy-methyl-glycinate (NIG) with disodium-ethylene diamine tetra-acetate (EDTA), with other known and widely used eye-drops preservatives. MATERIALS AND METHODS: Rabbit corneal cells (SIRC) were tested either in 96-well plates or in suspension culture. Treatments with preservatives (used at known bacteriostatic concentrations) included: benzalkonium chloride (BAK), polyquaternium-1 (PQ-1), sodium perborate (SP: NaBO3 * H2O), and NIG ± EDTA at different concentrations (0.001% and 0.002%), and different treatment times (from 30 minutes to 120 hours). At the end of treatment, cell survival was evaluated by a specific spectrophotometric method through the metabolic conversion of MTT [3-(4,5-dimethylthiazol-2-yl) 2, 5-diphenyltetrazolium bromide] into formazan crystals. RESULTS: Almost no cell toxicity was evident for NIG and SP at either concentration (0.001% or 0.002%), while a low toxicity was observed for PQ-1 (62% at the highest dose at 120 hours). BAK, as expected, showed the highest toxicity (60-80% at 30 minutes, and over 90% from eight hours onward). EDTA 0.1% alone or in combination with NIG 0.002%, showed no toxicity at 24 hours, and even resulted in cell growth promotion (46% and 38%, respectively), after 48 hours of treatment. CONCLUSIONS: These data show that the new preservative NIG/EDTA, at doses known to have effective antimicrobial properties, has a very low toxicity on corneal cells, and so it can be safely used in multi-dose eye drops.

Melanogenesis in uveal melanoma cells: Effect of argan oil.

The mechanisms underlying cutaneous melanogenesis have been widely studied; however, very little is known about uveal melanogenesis. Melanin is normally produced by uveal melanocytes and gives the color to the iris. A derangement from this normal production may occur, for instance, by iatrogenic events, such as glaucoma therapy with prostaglandins that may enhance cutaneous and iris pigmentation. In this study, we investigated the mechanisms that regulate uveal melanogenesis in human uveal melanoma cells (92.1) and murine cutaneous melanoma cells (B16-F1). In the first part of the study, we compared the effects of known cutaneous pigmenting agents on the B16-F1 and 92.1 cells, showing an opposite response of the two cell lines. Subsequently, using argan oil, a known depigmenting agent for murine cutaneous melanoma cells, on 92.1 cells, we found that in these cells, it also functioned as an inhibitor of melanogenesis and tyrosinase expression. From a molecular perspective, treatment of the 92.1 cells with argan oil decreased melanogenesis-associated transcription factor (MITF) gene expression by inducing MITF phosphorylation at Ser73, thus leading to MITF ubiquitination and disposal. It also led to the downregulation of the extracellular signal-regulated kinase (ERK)1/2 and Akt pathways, also known to be involved in cutaneous melanogenesis, although with an opposing function. Taken together, our data indicate that: ⅰ) some differences exist in the regulation of melanogenesis between cutaneous and uveal melanoma cells; and ⅱ) argan oil exerts a depigmenting effect on 92.1 cells through its action on the ERK1/2 and Akt pathways.

Classical VEGF, Notch and Ang signalling in cancer angiogenesis, alternative approaches and future directions (Review).

Angiogenesis is the formation of new vessels starting from pre-existing vasculature. Tumour environment is characterized by 'aberrant angiogenesis', whose main features are tortuous and permeable blood vessels, heterogeneous both in their structure and in efficiency of perfusion and very different from normal vessels. Therapeutic strategies targeting the three pathways chiefly involved in tumour angiogenesis, VEGF, Notch and Ang signalling, have been identified to block the vascular supply to the tumour. However, phenomena of toxicity, development of primary and secondary resistance and hypoxia significantly blunted the effects of anti-angiogenic drugs in several tumour types. Thus, different strategies aimed to overcome these problems are imperative. The focus of the present review was some principal 'alternative' approaches to classic antiangiogenic therapies, including the cyclooxygenase-2 (COX-2) blockade, the use of oligonucleotide complementary to the miRNA to compete with the mRNA target (antimiRs) and the inhibition of matrix metalloproteinases (MMPs). The role of blood soluble VEGFA as a predictive biomarker during antiangiogenic therapy in gastric, ovarian and colorectal cancer was also examined.

Gabapentin Attenuates Ocular Inflammation: In vitro and In vivo Studies.

To investigate the effects of gabapentin, a structural analog of γ-amino butyric acid (GABA), on the inflammatory response of lipopolysaccharide (LPS)-stimulated rabbit corneal cells (SIRC) and on endotoxin-induced uveitis (EIU) in rabbits. We investigated the LPS-induced expression of several inflammatory mediators, such as TNF-α, IL-1ß, cPLA2, COX-2, and PGE2 in the SIRC cells with or without gabapentin treatment. Gabapentin treatment significantly (p < 0.05) attenuated cytokines production, cPLA2 activation, COX-2 expression, and PGE2 levels in SIRC. EIU was induced by an intraocular injection of 0.1 µg of LPS in albino rabbit eye. After 7 and 24 h from LPS injection clinical signs of ocular inflammation were examined by slit lamp with or without topical treatment of 0.5% gabapentin. Tears, aqueous, cornea, conjunctiva, and iris-ciliary body were collected and inflammatory biomarkers assessed. Topical treatment with gabapentin significantly (p < 0.05) reduced clinical signs and biomarkers of inflammation compared with the LPS group both at 7 and 24 h. In conclusion, the results generated in the present study suggest that ophthalmic formulation based on gabapentin may be useful in the treatment of inflammatory conditions associated to ocular pain such as uveitis, and that clinical studies to evaluate this possibility may be warranted.

Molecular Mechanisms Mediating Antiangiogenic Action of the Urokinase Receptor-Derived Peptide UPARANT in Human Retinal Endothelial Cells.

PURPOSE: To investigate the molecular mechanisms of the antiangiogenic activity of UPARANT, an antagonist of the urokinase-type plasminogen activator receptor (uPAR), on primary human retinal endothelial cells (HREC) as a model of in vitro angiogenesis. METHODS: The antiangiogenic activity of UPARANT was evaluated on endothelial cell migration, invasion, and tube formation. Human REC were further analyzed for viability, transendothelial electrical resistance (TEER), and tight junction (TJ) expression at the protein and mRNA levels. Vascular endothelial growth factor-related signaling molecules were also analyzed by Western and northern blots. RESULTS: UPARANT inhibited in a dose-dependent fashion HREC motility, invasion, and tube formation stimulated by VEGF-A, in a range of doses (1-100 nM) that had no effect on cell viability and proliferation. UPARANT also prevented the loss of permeability induced by VEGF-A, restoring normal TEER values and TJ protein expression. At the molecular level, UPARANT inhibited VEGFR-2 and STAT3 phosphorylation, thus decreasing VEGF and hypoxia-inducible factor 1-alpha expression, finally resulting in decreased activation of MEK/ERK, JNK, p38, and AKT signaling proteins. CONCLUSIONS: These findings indicate that UPARANT exerts its antiangiogenic effects through the inhibition of the downstream signaling activated by angiogenic factors such as VEGF-A.