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This work presents the development and validation of a quantitative HILIC UHPLC-ESI-QTOF-MS/MS method for amino acids combined with untargeted metabolic profiling of human corneal epithelial (HCE) cells after treatment with ionic liquids. The work included a preliminary metabotoxicity screening of 14 different ionic liquids, of which 9 carefully selected ionic liquids were chosen for a metabolomics study. This study is focused on the correlation between the toxicity of the ionic liquids and their metabolic profiles. The method development included the comparison of different MS/MS acquisition modes. A sequential window acquisition of all theoretical fragment ion mass spectra (SWATH) method with variable Q1 window widths and narrow Q1 target windows of 5 Da for most of the amino acids was selected as the optimal acquisition mode. Due to the absence of a true blank matrix, C-13,N-15-isotopically labelled amino acids were utilized as surrogate calibrants, instead of proteinogenic amino acids. Partial least squares (PLS) analysis of the median effective concentrations (EC50) of 9 selected ionic liquids showed a correlation with their metabolic profile measured by the untargeted screening.

PURPOSE: Matrix regenerating agents (RGTAs) emerged as promising in vivo wound-healing agents. These agents could prove beneficial for the treatment of dry eye disease-associated corneal micro-erosions; therefore, we aimed to evaluate the wound healing efficacy of regenerative agents (RGTAs or serum) in an in vitro model of hyperosmolarity (HO) stressed and non-stressed human corneal epithelial cells. PATIENTS AND METHODS: The migration and proliferation induced by the regenerative agents was evaluated using an in vitro scratch wound assay and brome-deoxy-uridine incorporation. The inflammatory profile and effects of osmoregulators were also investigated. The two-tailed paired t-test calculated the statistical significance, with P-value

Purpose Most pure glaucoma drugs (pGDs) are hydrophobic substances intended to reduce elevated intraocular pressure. The aims of our study were to determine the toxicity of pGDs (brimonidine tartrate, brinzolamide, latanoprost, timolol maleate, and pilocarpine hydrochloride) on ocular surface cells and to establish whether their toxicity is subsequent to cellular membrane destabilization. Methods The toxicity of clinically efficient doses of pGDs was measured at different time points in a cell culture of human corneal epithelial cells using a redox indicator. pGD interaction with the plasma membrane was analyzed using a hemolysis assay and liposome electrokinetic chromatography. The capacity of pGDs to induce endoplasmic reticulum stress was investigated by immunoblotting. Results The toxicity assay showed that all pGDs decrease the viability of the epithelial cells to variable degrees. Early toxicity was measured for 4% pilocarpine and 0.15% brimonidine with 60% cell death at 4 hours, whereas 2% pilocarpine and 0.005% latanoprost showed almost 100% toxicity but only after 16 hours. The hemolysis assay and liposome electrokinetic chromatography experiments suggested that interaction between pGDs and lipid membranes is weak and cannot explain cell death through lysis. Immunoblotting revealed that the drugs activate endoplasmic reticulum stress and, with the exception of pilocarpine, have the capacity to induce apoptosis through upregulation of C/EBP homologous protein. Conclusions Our study indicates that all studied pGDs decrease the viability of the corneal epithelial cells, but none of the tested compounds were able to destabilize cellular membranes. The pGDs seem to be internalized and can induce apoptosis through C/EBP homologous protein recruitment.

Purpose Treating corneal neurotrophic ulcers is challenging. Topical application of matrix regeneration therapy (RGTA), which is a dextran derivative polymer and heparan sulfate analog, is a promising regenerative therapy and an alternative or additional therapeutic regimen when corneal healing is compromised. The aim of this study was to evaluate the efficacy of RGTA (Cacicol) in the treatment of 6 patients with severe neurotrophic ulcers. Methods We present an uncontrolled prospective case series of 6 patients (6 eyes) with severe corneal neurotrophic ulcers. Patients were treated with topical RGTA at a dose of 1 drop every second day. The main outcome measure was complete corneal epithelialization. We measured corneal thickness by anterior segment swept-source optical coherence tomography. Results Two patients (33%) showed complete corneal healing, one at 6 weeks and the other at 10 weeks. Treatment was considered failure in 4 patients (67%), and 1 patient had corneal perforation. None of the patients showed improvement in best-corrected visual acuity. There were no RGTA-related local or systemic side effects. Conclusions Based on previous studies, RGTA seems to be a promising therapeutic agent for controlling ocular surface inflammation and promoting corneal healing. In this study, the efficacy of RGTA did not match the encouraging results from previous reports.

This study aims at extending the understanding of the toxicity mechanism of ionic liquids (ILs) using various analytical methods and cytotoxicity assays. The cytotoxicity of eight ILs and one zwitterionic compound was determined using mammalian and bacterial cells. The time dependency of the IL toxicity was assessed using human corneal epithelial cells. Hemolysis was performed using human red blood cells and the results were compared with destabilization data of synthetic liposomes upon addition of ILs. The effect of the ILs on the size and zeta potential of liposomes revealed information on changes in the lipid bilayer. Differential scanning calorimetry was used to study the penetration of the ILs into the lipid bilayer. Pulsed field gradient nuclear magnetic resonance spectroscopy was used to determine whether the ILs occurred as unimers, micelles, or if they were bound to liposomes. The results show that the investigated ILs can be divided into three groups based on the cytotoxicity mechanism: cell wall disrupting ILs, ILs exerting toxicity through both cell wall penetration and metabolic alteration, and ILs affecting solely on cell metabolism.

The toxicity of some promising biomass-dissolving amidinium-, imidazolium-, and phosphonium-based ionic liquids (ILs), toward two different cell lines, human corneal epithelial cells and Escherichia coli bacterial cells, was investigated. In addition, dynamic light scattering (DLS) and ζ potential measurements were used to study the effect of the ILs on the size and surface charge of some model liposomes. Capillary electrophoresis (CE) was used for determination of the electrophoretic mobilities of the liposomes and for determination of the critical micelle concentration (cmc) of the ILs. The toxicity of the phosphonium ILs was highly dependent on the longest linear chain of the IL, due to increasing hydrophobicity, with the long-chain phosphonium ILs being toxic while the shorter-chain versions were significantly less toxic or not toxic at all. Amidinium and imidazolium ILs showed no significant effect on the cells, within the concentration range used. Moreover, the more hydrophobic ILs were found to have a major effect on the surface charges and size distributions of the model liposomes, which can lead to disruption of the lipid bilayer. This indicates that the cytotoxicity is at least to some extent dependent on direct interactions between ILs and the biomembrane.

To explore factors related to pathogenesis of rhegmatogenous retinal detachment (RRD) and development of proliferative vitreoretinopathy (PVR), vitreous levels of angiopoietin-1 and -2 (Ang-1 and -2), previously undefined in RRD, transforming growth factor-(TGF) β1, vascular endothelial growth factor (VEGF), erythropoietin (EPO) and proteolytic mediators of extracellular matrix remodelling (MMP-2 and -9) were compared in eyes with RRD and eyes with idiopathic macular hole or pucker.Vitreous samples were collected from 117 eyes with RRD (study group) and 40 eyes with macular hole or pucker (control group). Growth factors were measured by ELISA and matrix metalloproteinases (MMPs) by gelatin zymography.The mean vitreous concentrations of Ang-2, MMP-2, and MMP-9 were higher (all p 0.01), whereas concentration of VEGF was lower (p = 0.01) in eyes with RRD relative to controls. Logistic regression analysis identified Ang-2 concentration as a novel marker of RRD (p = 0.0001, OR 48.7). Ang-1, EPO, and total TGF-β1 levels were not significantly different between the groups. However, TGF-β1 and MMP-2 were increased in eyes with total RRD compared to those with local RRD (p ≤ 0.05). In eyes with PVR, no differences were observed in any studied marker as compared with non-PVR eyes.Current results reveal Ang-2 as a key factor upregulated in RRD. It may co-operate with fibrosis-associated factors and contribute to vascular complications such as breakdown of blood-eye barrier and PVR development.

Purpose: Angiogenesis in diabetic retinopathy (DR) is a multifactorial process regulated by hypoxia-induced growth factors and inflammatory cytokines. In addition to the angiogenic switch, the proteolytic processing and altered synthesis of the extracellular matrix are critical steps in this disease. This study was performed to evaluate the levels of matrix metalloproteinase-2 and matrix metalloproteinase-9 (MMP-2 and MMP-9), angiopoietin-1 and angiopoietin-2 (Ang-1 and Ang-2), vascular endothelial growth factor (VEGF), erythropoietin (EPO) and transforming growth factor-β1 (totalTGFβ1) in the vitreous of diabetic eyes undergoing vitrectomy compared with control eyes operated because of macular hole or pucker. Methods: Prospective consecutive controlled observational study performed in the unit of vitreoretinal surgery in Finland during the years 2006–2008. Vitreous samples were collected before the start of the conventional 3-ppp vitrectomy. Vitreous MMP-2 and MMP-9, Ang-1 and Ang-2, VEGF, EPO and TGFβ1 concentrations were measured from 69 patients with Type 1 or 2 diabetes and 40 controls. Results: Comparison of eyes with DR with controls revealed that the mean vitreous concentrations of proMMP-2 (p = 0.0015), totalMMP-2 (p = 0.0011), proMMP-9 (p = 0.00001), totalMMP-9 (p < 0.00001), Ang-2 (p < 0.00001), VEGF (p < 0.00001), EPO (p < 0.00001) and totalTGFβ1 (p = 0.000026) were significantly higher in the former group. A multivariate logistic regression analysis suggested intravitreal Ang-2 concentration being the key marker of PDR (p = 0.00025) (OR = 1507.9). Conclusion: The main new finding is that the intravitreal concentrations of Ang-2 correlated significantly with MMP-9, VEGF, EPO and TGFβ1 levels in diabetic eyes undergoing vitrectomy. Thus, these factors could promote retinal angiogenesis synergistically.

Hyperosmolarity (HO) imposes a remarkable stress on membranes, especially in tissues in direct contact with the external environment. Our efforts were focused on revealing stress-induced lipid changes that precede the inflammatory cytokine response in human corneal epithelial cells exposed to increasing osmolarity. We used a lipidomic analysis that detected significant and systematic changes in the lipid profile, highly correlated with sodium concentrations in the medium. Ceramides and triglycerides (TGs) were the most-responsive lipid classes, with gradual increases of up to 2- and 3-fold, respectively, when compared with control. The source of ceramide proved to be sphingomyelin hydrolysis, and neutral sphingomyelinase 2 (NSM2) activity showed a 2-fold increase 1 h after HO stress, whereas transcription increased 3-fold. Both TG accumulation and IL-8 secretion were shown to be dependent on ceramide production by specific knock-down of NSM2. In HCE cells, diglyceride acyltransferase 1 was responsible for the TG synthesis, but the enzyme activity had no effect on cytokine secretion. Hence, NSM2 plays a key role in the cellular response to hyperosmolar stress, and its activity regulates both cytokine secretion and lipid droplet formation.

Lysosomal acid lipase (LAL), the only lysosomal enzyme involved in the hydrolysis of LDL-cholesteryl esters, is a key regulator of cellular cholesterol and fatty acid homeostasis and its deficiency contributes to the pathophysiology of various diseases. In this study, we questioned whether oxidized or glycated LDL, a common occurrence in atherosclerosis and diabetes, affect the activity and expression of LAL in vascular endothelial cells (EC) and smooth muscle cells (SMC). LAL activity and expression were assayed in cultured human EC and SMC exposed to oxidized LDL (oxLDL), (±)9-hydroxyoctadecadienoic acid-cholesteryl ester (HODE), glycated LDL (gLDL), or native LDL (nLDL) as control, in the presence or absence of LXR or PPAR-gamma agonists. We found that LAL activity and expression were significantly down regulated by oxLDL and HODE in EC, and by gLDL in SMC. The LXR agonist T0901317 reversed the decreased LAL expression in modified LDL- or HODE-exposed EC (P

In addition to circulation, where it transfers phospholipids between lipoprotein particles, phospholipid transfer protein (PLTP) was also identified as a component of normal tear fluid. The purpose of this study was to clarify the secretion route of tear fluid PLTP and elucidate possible interactions between PLTP and other tear fluid proteins. Human lacrimal gland samples were stained with monoclonal antibodies against PLTP. Heparin-Sepharose (H-S) affinity chromatography was used for specific PLTP binding, and coeluted proteins were identified with MALDI-TOF mass spectrometry or Western blot analysis. Immunoprecipitation assay and blotting with specific antibodies helped to identify and characterize PLTP-mucin interaction in tear fluid. Human tear fluid PLTP is secreted from the lacrimal gland. MALDI-TOF analysis of H-S fractions identified several candidate proteins, but protein-protein interaction assays revealed only ocular mucins as PLTP interaction partners. We suggest a dual role for PLTP in human tear fluid: (1) to scavenge lipophilic substances from ocular mucins and (2) to maintain the stability of the anterior tear lipid film. PLTP may also play a role in the development of ocular surface disease.

Purpose Since homeostasis at the ocular surface requires a delicate balance between numerous factors, and the external environment contributes as an unpredictable component, we aimed to understand the role that various lipids and their regulators have in the complex process that maintains a healthy corneal surface. Methods Through basic proteomics, we tested the presence of sphingolipid metabolism enzymes in normal human tears, and then used a cell culture model to study how the proteins are secreted and for what purpose. Results When studying healthy tears, we found that sphingolipid-specific enzymes, acid and neutral sphingomyelinases, and ceramidases can be detected. The role played by sphingolipid metabolism in stress provided the motivation for further studies concerning their secretion/leakage in the extracellular environment in a cell culture model of human corneal epithelial cells (HCE). Among the stress agents investigated (i.e., ultraviolet B [UV-B] radiation, hyperosmolarity [HO], and lipopolysaccharide [LPS]), UV-B and HO induced dose-dependent release/secretion of sphingomyelinases from the cells. In an attempt to identify the route of secretion or release of the enzyme, we discovered that the tested stress stimuli induced shedding of extracellular vesicles in the HCE-conditioned medium. Conclusions Extracellular stress affects tear fluid composition more profoundly than just secretion of proinflammatory mediators. Lipids at the ocular surface, either in tear fluid or within the corneal epithelial cells, can be modified by a relatively large array of lipases to modulate their functions. Moreover, extracellular vesicles in the tear fluid could represent a valuable noninvasive diagnosis tool for anterior segment diseases.

Purpose: Sphingolipid (SL) research reached a peak in the past years. Yet this positive trend was not evident for eye research as the relative number of studies centered on SLs is decreasing. Our aim is to encourage the inclusion of SL metabolites in studies of ocular pathophysiology by summarizing recent findings and current awareness concerning ceramides in the anterior segment of the eye. Methods: Review of literature relating to ceramides as bioactive lipids and the extent to which their particular nature was investigated in ocular pathophysiology. Results: Ceramides are rare but indispensable lipids that influence cellular responses through their effects on membrane biophysical properties or direct interaction with target proteins. Their biological significance is increased by variability and adaptability as there are tens of enzymes designed to modulate their function. The eye offers a set of unique environments where ceramides or other SLs have not been extensively studied. Not surprisingly, ceramides were associated with apoptosis in the metabolically active tissues, while little is known about its effects on the biophysical properties of the tears or lens lipids. More so, there are still aspects of the ocular homeostasis control where SLs contribution has not been investigated to date (e.g. pathogen aggression). Conclusions: Ceramides and SL metabolism still receive increasing attention and have proven to be a significant metabolite in many research fields (e.g. cancer, stress response and inflammation) and there are yet many questions that they will aid answer. With the present work, we seek to increase awareness of these lipids also in eye research and to highlight their importance as common regulators of various diseases.

PURPOSE. Climatic droplet keratopathy (CDK) is a degenerative disease of the cornea with possible involvement from matrix metalloproteinases (MMPs). Therefore, the authors investigated histologic distribution, levels, and molecular forms of MMP- 2 and MMP-9, as well as tear fluid levels of MMPs and cytokines in CDK patients. They additionally examined UV-B-irradiation effect on production of gelatinases and cytokines by human corneal epithelial (HCE) cell culture model. METHODS. Tears were collected from 20 unrelated individuals (10 with CDK and 10 controls). CDK affected corneas were haematoxylin-eosin stained and the presence and distribution of MMP-2 and MMP-9 was examined using immunohistochemistry. Gelatinases and cytokine secretion was measured in tears and supernatants from UV-B-exposed HCEs by immunoblotting, gelatin zymography, and protein array, respectively. RESULTS. MMP-2 and MMP-9 values were significantly higher in tears collected from CDK patients than healthy controls and were accompanied by pro-inflammatory cytokine secretion. Immunohistochemistry showed that MMP-2 was expressed at the basement membrane zone in both control and affected corneas, but also marked the edges of the granular CDK deposits; MMP-9 expression was restrained to basal layers of the epithelium and was markedly induced in CDK corneas. In HCE cells, UV-B increased gelatinase secretion, with a striking effect on MMP-9, and was preceded by pro-inflammatory cytokine release. CONCLUSIONS. The authors demonstrate that the corneal epithelium could participate in CDK development as a source of cytokines and gelatinases. Additionally, in HCE cells, UV-B- modulated cytokine and subsequent MMP secretion. Local inhibition of cytokine secretion and gelatinases may prevent CDK progression. © 2012 The Association for Research in Vision and Ophthalmology, Inc. Fil: Holopainen, Juha M.. University Of Helsinski; Finlandia Fil: Robciuc, Alexandra. University Of Helsinski; Finlandia Fil: Cafaro, Thamara Analia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina Fil: Suarez, Maria Fernanda. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina Fil: Konttinen, Yrjö T.. University Of Helsinski; Finlandia Fil: Alkatan, Hind M.. The Eye Center and The Eye Foundation for Research in Ophthalmology; Arabia Saudita Fil: Tabbara, Khalid F.. The Eye Center and The Eye Foundation for Research in Ophthalmology; Arabia Saudita Fil: Tervahartiala, Taina. University Of Helsinski; Finlandia Fil: Sorsa, Timo. University Of Helsinski; Finlandia Fil: Urrets Zavalía, Julio Alberto. Universidad Catolica de Córdoba. Facultad de Medicina. Clinica Universitaria Reina Fabiola. Dto de Oftalmologia; Argentina Fil: Serra, Horacio Marcelo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina

The study aim was to investigate NOS3 VNTR, NOS3 G894T, EDN1 C8002T, ACE I/D, AGT M235T and AGTR1 A1166C in nonobese and obese T2DM patients, and their interaction with the incidence of microangiopathy. T2DM subjects (n=250; 166 nonobese, and 84 obese) were genotyped for the gene variants by PCR/RFLP. The interaction of these polymorphisms with obesity and their contribution to microangiopathy were analyzed by multivariate regression analysis. A higher frequency of NOS3 4a allele was found in obese (P=0.027) vs. nonobese subjects. ACE D (P=0.009) and AGT 235T (P=0.026) alleles were associated with the reduced risk of diabetic nephropathy in nonobese and obese patients, respectively. In obese subjects, NOS3 4a (P=0.011) had a converse effect to NOS3 894T (P=0.043), and EDN1 8002T (P=0.035) on the prevalence of combined microangiopathy (neuropathy/retinopathy/nephropathy) vs. microangiopathy-negative subjects. The study indicates association of RAS variants with obesity and nephropathy, and an opposite effect of NOS3 VNTR and NOS3 G894T on the occurrence of combined microangiopathy.