Publications by authors named "Alexandra Robciuc"

16 Publications

  • Page 1 of 1

A combined targeted/untargeted LC-MS/MS-based screening approach for mammalian cell lines treated with ionic liquids: Toxicity correlates with metabolic profile.

Talanta 2019 May 15;197:472-481. Epub 2019 Jan 15.

Institute of Pharmaceutical Sciences, Pharmaceutical (Bio-)Analysis, University of Tübingen, Auf der Morgenstelle 8, 72076 Tübingen, Germany. Electronic address:

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,N-isotopically labelled amino acids were utilized as surrogate calibrants, instead of proteinogenic amino acids. Partial least squares (PLS) analysis of the median effective concentrations (EC) of 9 selected ionic liquids showed a correlation with their metabolic profile measured by the untargeted screening.
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http://dx.doi.org/10.1016/j.talanta.2019.01.054DOI Listing
May 2019

Correlation between Ionic Liquid Cytotoxicity and Liposome-Ionic Liquid Interactions.

Chemistry 2018 Feb 24;24(11):2669-2680. Epub 2018 Jan 24.

Department of Chemistry, Faculty of Science, University of Helsinki, A.I. Virtasen aukio 1, 00560, Helsinki, Finland.

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.
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http://dx.doi.org/10.1002/chem.201704924DOI Listing
February 2018

Pure Glaucoma Drugs Are Toxic to Immortalized Human Corneal Epithelial Cells, but They Do Not Destabilize Lipid Membranes.

Cornea 2017 Oct;36(10):1249-1255

*Helsinki Eye Lab, Department of Ophthalmology, Helsinki University Hospital and University of Helsinki, Helsinki, Finland; ‡Genetics and Biomarkers Unit, National Institute of Health and Welfare, Helsinki, Finland; §Department of Chemistry, University of Helsinki, Helsinki, Finland; and ¶Department of Opthalmology, Faculty of Medicine, University François-Rabelais, Tours, France.

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.
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http://dx.doi.org/10.1097/ICO.0000000000001322DOI Listing
October 2017

Distribution of local anesthetics between aqueous and liposome phases.

J Chromatogr A 2017 Jan 3;1479:194-203. Epub 2016 Dec 3.

Department of Chemistry, POB 55, 00014 University of Helsinki, Finland. Electronic address:

Liposomes were used as biomimetic models in capillary electrokinetic chromatography (EKC) for the determination of distribution constants (K) of certain local anesthetics and a commonly used preservative. Synthetic liposomes comprised phosphatidylcholine and phosphatidylglycerol phospholipids with and without cholesterol. In addition, ghost liposomes made from red blood cell (RBC) lipid extracts were used as pseudostationary phase to acquire information on how the liposome composition affects the interactions between anesthetics and liposomes. These results were compared with theoretical distribution coefficients at pH 7.4. In addition to 25°C, the distribution constants were determined at 37 and 42°C to simulate physiological conditions. Moreover, the usability of five electroosmotic flow markers in liposome (LEKC) and micellar EKC (MEKC) was studied. LEKC was proven to be a convenient and fast technique for obtaining data about the distribution constants of local anesthetics between liposome and aqueous phase. RBC liposomes can be utilized for more representative model of cellular membranes, and the results indicate that the distribution constants of the anesthetics are greatly dependent on the used liposome composition and the amount of cholesterol, while the effect of temperature on the distribution constants is less significant.
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http://dx.doi.org/10.1016/j.chroma.2016.12.005DOI Listing
January 2017

Matrix Regeneration Therapy: A Case Series of Corneal Neurotrophic Ulcers.

Cornea 2016 Apr;35(4):451-5

*Helsinki Eye Lab, Department of Ophthalmology, University of Helsinki, Helsinki University Hospital, Helsinki, Finland; and †Genomics and Biomarkers Unit, National Institute for Health and Welfare, Helsinki, Finland.

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.
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http://dx.doi.org/10.1097/ICO.0000000000000759DOI Listing
April 2016

Impact of amphiphilic biomass-dissolving ionic liquids on biological cells and liposomes.

Environ Sci Technol 2015 Feb 21;49(3):1870-8. Epub 2015 Jan 21.

Department of Chemistry and ∥Laboratory of Organic Chemistry, Department of Chemistry, University of Helsinki , A. I. Virtasen Aukio 1, Post Office Box 55, FIN-00014 University of Helsinki, Finland.

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.
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http://dx.doi.org/10.1021/es505725gDOI Listing
February 2015

Lipid-modifying enzymes in human tear fluid and corneal epithelial stress response.

Invest Ophthalmol Vis Sci 2014 Jan 2;55(1):16-24. Epub 2014 Jan 2.

Helsinki Eye Lab, Department of Ophthalmology, University of Helsinki, Helsinki, Finland.

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.
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http://dx.doi.org/10.1167/iovs.13-12577DOI Listing
January 2014

Increased intravitreal angiopoietin-2 levels associated with rhegmatogenous retinal detachment.

Graefes Arch Clin Exp Ophthalmol 2014 Jun 12;252(6):881-8. Epub 2013 Nov 12.

Unit of Vitreoretinal Surgery, Department of Ophthalmology, Helsinki University Central Hospital, Haartmaninkatu 4 C, 00290, Helsinki, Finland,

Purpose: 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.

Methods: 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.

Results: 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.

Conclusions: 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.
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http://dx.doi.org/10.1007/s00417-013-2508-zDOI Listing
June 2014

Ceramides in the pathophysiology of the anterior segment of the eye.

Curr Eye Res 2013 Oct 25;38(10):1006-16. Epub 2013 Jul 25.

Department of Ophthalmology, University of Helsinki, Helsinki Eye Lab, Helsinki, Finland.

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.
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http://dx.doi.org/10.3109/02713683.2013.810273DOI Listing
October 2013

Ang-2 upregulation correlates with increased levels of MMP-9, VEGF, EPO and TGFβ1 in diabetic eyes undergoing vitrectomy.

Acta Ophthalmol 2013 Sep 26;91(6):531-9. Epub 2012 Oct 26.

Unit of Vitreoretinal Surgery, Department of Ophthalmology, Helsinki University Central Hospital, Finland.

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.
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http://dx.doi.org/10.1111/j.1755-3768.2012.02473.xDOI Listing
September 2013

Hyperosmolarity-induced lipid droplet formation depends on ceramide production by neutral sphingomyelinase 2.

J Lipid Res 2012 Nov 15;53(11):2286-95. Epub 2012 Aug 15.

Helsinki Eye Lab, Department of Ophthalmology, Haartmaninkatu 4 C, University of Helsinki, 00290 Helsinki, Finland.

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.
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http://dx.doi.org/10.1194/jlr.M026732DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3465998PMC
November 2012

Chromatographic lipid profiling of stress-exposed cells.

J Sep Sci 2012 Aug 24;35(15):1845-53. Epub 2012 May 24.

Laboratory of Analytical Chemistry, Department of Chemistry, University of Helsinki, Helsinki, Finland.

Lipidomics is an emerging field of science not only due to its integral part of cell biology and biophysics but also due to the key role of lipids in the modulation of membrane physical properties, signaling, and cell death regulation. The aim of this study was to characterize changes in N-palmitoyl ceramide concentration and in the global lipid profile in macrophages challenged by oxidized low-density lipoprotein and nutrient deprived hepatocytes. For this purpose, a quantitative targeted method based on gas chromatography-mass spectrometry for the determination of total N-palmitoyl ceramide concentrations in the cellular membranes of cells under stress was used. Ultrahigh-performance liquid chromatography-quadrupole-time of flight mass spectrometry was applied for the comprehensive profiling of lipids. In essence, we found that both models of cellular stress caused an increase in N-palmitoyl ceramide levels. In addition, increased levels of other ceramides were observed as well as up- and down-regulation of several other lipid species.
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http://dx.doi.org/10.1002/jssc.201200252DOI Listing
August 2012

Pro-inflammatory cytokines and gelatinases in climatic droplet keratopathy.

Invest Ophthalmol Vis Sci 2012 Jun 14;53(7):3527-35. Epub 2012 Jun 14.

Helsinki Eye Lab, Department of Ophthalmology, University of Helsinki, Finland.

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.
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http://dx.doi.org/10.1167/iovs.12-9441DOI Listing
June 2012

Modified low density lipoproteins decrease the activity and expression of lysosomal acid lipase in human endothelial and smooth muscle cells.

Cell Biochem Biophys 2011 Sep;61(1):209-16

Institute of Cellular Biology and Pathology N. Simionescu, 8, B. P. Hasdeu Street, PO Box 14-35, 050568 Bucharest, Romania.

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 < 0.001) and in gLDL-exposed SMC, whereas PPAR-gamma agonist rosiglitazone induced a low effect only in EC. In conclusion, modified LDL down regulates LAL expression in human EC and SMC by a process involving the LXR signaling pathway. This is the first demonstration that modified LDL modulate LAL expression, in a cell specific manner.
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http://dx.doi.org/10.1007/s12013-011-9190-8DOI Listing
September 2011

Identification of gene variants in NOS3, ET-1 and RAS that confer risk and protection against microangiopathy in type 2 diabetic obese subjects.

Biochem Biophys Res Commun 2011 Apr 21;407(3):486-90. Epub 2011 Mar 21.

Institute of Cellular Biology and Pathology N. Simionescu, 8, B.P. Hasdeu Street, 050568 Bucharest, Romania.

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.
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http://dx.doi.org/10.1016/j.bbrc.2011.03.043DOI Listing
April 2011

Interaction of phospholipid transfer protein with human tear fluid mucins.

J Lipid Res 2010 Nov 19;51(11):3126-34. Epub 2010 Aug 19.

Department of Ophthalmology, University of Helsinki, Finland.

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.
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http://dx.doi.org/10.1194/jlr.M006239DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2952553PMC
November 2010