Molecular and cellular effects of ultraviolet light-induced genotoxicity

Exposure to the solar ultraviolet spectrum that penetrates the Earth's stratosphere (UVA and UVB) causes cellular DNA damage within skin cells. This damage is elicited directly through absorption of energy (UVB), and indirectly through intermediates such as sensitizer radicals and reactive oxygen species (UVA). DNA damage is detected as strand breaks or as base lesions, the most common lesions being 8-hydroxydeoxyguanosine (8OHdG) from UVA exposure and cyclobutane pyrimidine dimers from UVB exposure. The presence of these products in the genome may cause misreading and misreplication. Cells are protected by free radical scavengers that remove potentially mutagenic radical intermediates. In addition, the glutathione-S-transferase family can catalyze the removal of epoxides and peroxides. An extensive repair capacity exists for removing (1) strand breaks, (2) small base modifications (8OHdG), and (3) bulky lesions (cyclobutane pyrimidine dimers). UV also stimulates the cell to produce early response genes that activate a cascade of signaling molecules (e.g., protein kinases) and protective enzymes (e.g., haem oxygenase). The cell cycle is restricted via p53-dependent and -independent pathways to facilitate repair processes prior to replication and division. Failure to rescue the cell from replication block will ultimately lead to cell death, and apoptosis may be induced. The implications for UV-induced genotoxicity in disease are considered.

Physiological effects of oxidized phospholipids and their cellular signaling mechanisms in inflammation

Oxidized phospholipids, such as the products of the oxidation of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine by nonenzymatic radical attack, are known to be formed in a number of inflammatory diseases. Interest in the bioactivity and signaling functions of these compounds has increased enormously, with many studies using cultured immortalized and primary cells, tissues, and animals to understand their roles in disease pathology. Initially, oxidized phospholipids were viewed largely as culprits, in line with observations that they have proinflammatory effects, enhancing inflammatory cytokine production, cell adhesion and migration, proliferation, apoptosis, and necrosis, especially in vascular endothelial cells, macrophages, and smooth muscle cells. However, evidence has emerged that these compounds also have protective effects in some situations and cell types; a notable example is their ability to interfere with signaling by certain Toll-like receptors (TLRs) induced by microbial products that normally leads to inflammation. They also have protective effects via the stimulation of small GTPases and induce up-regulation of antioxidant enzymes and cytoskeletal rearrangements that improve endothelial barrier function. Oxidized phospholipids interact with several cellular receptors, including scavenger receptors, platelet-activating factor receptors, peroxisome proliferator-activated receptors, and TLRs. The various and sometimes contradictory effects that have been observed for oxidized phospholipids depend on their concentration, their specific structure, and the cell type investigated. Nevertheless, the underlying molecular mechanisms by which oxidized phospholipids exert their effects in various pathologies are similar. Although our understanding of the actions and mechanisms of these mediators has advanced substantially, many questions do remain about their precise interactions with components of cell signaling pathways.

Characterization and effects on cAMP accumulation of adrenomedullin and calcitonin gene-related peptide (CGRP) receptors in dissociated rat spinal cord cell culture

1 Adrenomedullin (AM) and calcitonin gene-related peptide (CGRP) have structural similarities, interact with each others receptors (calcitonin receptor-like receptor (CLR)/receptor-activity-modifying proteins (RAMPs)) and show overlapping biological activities. AM and CGRP receptors are chiefly coupled to cAMP production. In this study, a method of primary dissociated cell culture was used to investigate the presence of AM and CGRP receptors and their effects on cAMP production in embryonic spinal cord cells. 2 Both neuronal and non-neuronal CLR immunopositive cells were present in our model. 3 High affinity, specific [ 125I]-AM binding sites (K(d) 79±9 pM and B(max) 571±34 fmol mg -1 protein) were more abundant than specific [ 125I]-CGRP binding sites (K(d) 12±0.7 pM and B(max) 32±2 fmol mg -1 protein) in embryonic spinal cord cells. 4 Specific [ 125I]-AM binding was competed by related molecules with a ligand selectivity profile of rAM>hAM(22-52)>rCGRPα>CGRP(8-37) ≫[r-(r*,s*)]-N-[2-[[5-amino-1-[[4-(4-pyridinyl)-1-piperazinyl] carbonyl]pentyl]amino]-1-[(3,5-dibromo-4-hydroxyphenyl)methyl]-2-oxoethyl]-4-(1, 4-dihydro-2-oxo-3(2H)-quinazolinyl)-,1-piperidinecarboxamide (BIBN4096BS). 5 Specific [ 125I]-CGRP binding was competed by rCGRPα>rAM≥ CGRP(8-37)≥BIBN4096BS>hAM(22-52). 6 Cellular levels of cAMP were increased by AM (pEC"5"0 10.2±0.2) and less potently by rCGRPα (pEC"5"0 8.9±0.4). rCGRPα-induced cAMP accumulation was effectively inhibited by CGRP(8-37) (pA"2 7.63±0.44) and hAM(22-52) (pA"2 6.18±0.21) while AM-stimulation of cAMP levels was inhibited by CGRP(8-37) (pA"2 7.41±0.15) and AM(22-52) (pA"2 7.26±0.18). BIBN4096BS only antagonized the effects of CGRP (pA"2 8.40±0.30) on cAMP accumulation. 7 These pharmacological profiles suggest that effects of CGRP are mediated by the CGRP"1 (CLR/RAMP1) receptor in our model while those of AM are related to the activation of the AM"1 (CLR/RAMP2) receptor subtype. © 2006 Nature Publishing Group All rights reserved.

Cellular interaction with novel biomaterials

The objective of this thesis is to report the behaviour of mammalian cells with biocompatible synthetic polymers with potential for applications to the human body. Composite hydrogel materials were tested as possible keratoprosthetic devices. It was found that surface topography is an important consideration, pores, channels and fibres exposed on the surface of the hydrogels tested can have significant effects on the extent of cell adheson and proliferation. It is recommended that the core component is fabricated out of one of the following to provide a non cell adhesive base; A8, A11, A13, A22, A23. The haptic periphery fabricated out of one of the following would provide a cell adhesive composite; A16, A30, A33, A37, A38, A42, A43, A44. The presence of vitronectin in the ocular tissue appears to lead to higher cell adhesion to the posterior surface of a contact lens when compared to the anterior surface. Group IV contact lenses adhere more cells than Group II contact lenses - this may indicate that more protein (including vitronectin) is able to adhere to the contact lens due to the Group IV contact lenses high water content and ionic hydrogel matrix. Artificial lung surfactant analogues were found to be non cytotoxic but also decreased cell proliferation when tested at higher concentrations. Poly(lysine ethyl ester adipamide) [PLETESA] had the most favourable response on cell proliferation and commercial styrene/maleic anhydride (pMA/STY sp2) the most pronounced inhibitory response. The mode of action that decreases cell proliferation appears to be through membrane destabilization. Tissue culture well plates coated with PLETESA allowed cells to adhere in a concentration dependent manner, multilaminar liposomes possibly of PLETESA were observed in solution in PLETESA coated wells. Polyhydroxybutryate (PHB) and polyhydroxyvalerate (PHV) blends that contained hydroxyapatite were found to be the most cell adhesive material of those materials tested. The blends that were most susceptible to degradation adhered the most cells in initial stages of degradation. The initial slight increase in cell adhesion may be due to the increased rugosity of the material. As the degradation continued the number of cells adhering to the samples decreased, this may indicate that the polarity was inhibitory to cell adhesion during the later stages of degradation.

Cellular responses to potential biomaterials

The aim of this study was to systematically investigate the factors considered to be responsible for anchorage-dependent cell behaviour to determine which, if any, of these factors exerts greater influence. An efficient means of doing so is the in vitro fibroblast cell culture model. The interaction of fibroblasts with novel substrata gives information about how a biological system reacts to a foreign material. The may ultimately lead to the development of improved biomaterials. This interdisciplinary study combines the elements of surface characterisation and biological testing to determine the nature of the biomaterial/host interface. Polarity and surface charge were found to have an important influence on fibroblast adhesion to hydrogel polymers, by virtue of their water-structuring effects. The same factors were found to affect cell adhesion on undegraded PHB-HV copolymers and their blends with polysaccharides. On degraded PHB-HV copolymers, the degradation process itself played the greatest role in influencing cell response. Increasing surface charge and mechanical instability in these polymers inhibited cell adhesion. Based on the observations of hydrogels and PHB-copolymers a novel material, gel-spun PHB was designed for use as a wound scaffold. In vitro tests using human and mammalian fibroblasts accentuated the importance of polarity and surface charge in determining cellular response. The overall view of cellular behaviour on a broad spectrum of materials highlighted the effects that polarity and surface charge have on water-structuring, and how this affects interfacial conversion. In degradable systems, mechanical stability also plays an inportant role in determining anchorage-dependent cell behaviour.

The effects of nitrogen mustard on plasma membrane function

The antitumour bifunctional alkylating agent nitrogen mustard (HN2) inhibited the unidirectional influx of the potassium congener, 86 rubidium, into murine PC6A plasmacytoma cells and L1210 leukaemia cells. The proliferation of L1210 cells in vitro was characterised and shown to be sentitive to HN2. 86Rubidium influx into cells from rapidly-dividing cultures was more sensitive to inhibition by HN2 than that of cells from stationary cultures. Three components of unidirectional 86Rb+ & K+ influx into proliferating L1210 cells were identified pharmacologically: approximately 40% was active to the Na+ K+ ATPase inhibitor ouabain (10-3M), 40% was sensitive to the `loop' diuretics bumetanide (10-4M) and furosemide (10-3M) and the remainder was insensitive to both ouabain and furosemide. HN2 (10-5M) selectively inhibited the diuretic-sensitive component, which was entirely dependent upon extracellular Na+ and Cl- ions, and was presumed to represent Na+ K+ Cl- cotransport activity. The system did not mediate K+ /K+ exchange or unidirectional 86Rb+ efflux; accordingly, 86Rb+ efflux was insensitive to HN2. Inhibition of 86Rb & K+ influx by 10-5M HN2 was accompanied by approximately 35% of cell volume under isosmotic conditions; thus intracellular Na+ and K+ concentrations remained unchanged. These effects followed lethal damage to the cells but preceded actual cell death; other cellular functions were maintained including accumulation of cycloleucine, transmembrane potential, permeability to trypan blue, intracellular pH, total intracellular glutathione and calcium concentrations. No evidence was found that elevated cAMP levels or reduced ATP levels were involved in modulation of 86Rb+ & K+ influx. However, the Na+ - depedent transport of an amino acid was inhibited in a manner which appeared to be independent of 86Rb & K+ influx. An HN2-resistant L1210R cell line was also resistant to furosemide, and lacked a component of 86Rb+ & K+ influx which was sensitive to furosemide (10-3M). The results strongly suggest that the Na+ K+ Cl- costransporter of L1210 cells is a cellular target for HN2. This lesion is discussed with reference to the cytotoxic effects of the agent.

The Role of protein kinase C modulation in the antiproliferative effects of bistratene A, bryostatin 1 and phorbol esters

PKC-mediated signalling pathways are important in cell growth and differentiation, and aberrations in these pathways are implicated in tumourigenesis. The objective of this project was to clarify the link between cell growth inhibition and PKC modulation.The PKC activators bryostatin 1 and 12-0-tetradecanoylphorbol-13-acetate (TPA) inhibited growth in A549 and MCF-7 adenocarcinoma cells with great potency, and induced HL-60 leukaemia cell differentiation. Bistratene A affected these cells similarly. Experiments were conducted to test the hypotheses that bistratene A exerts its effects via PKC modulation and that characteristics of cytostasis induced by bryostatin 1 and TPA depend upon PKC isozyme-specific events. After incubation of A549 cells with TPA or bistratene A, 2D phosphoprotein electrophoretograrns revealed three proteins phosphorylated by both agents. However, bistratene A was unable to induce the formation of cellular networks on the basement membrane substitute Matrigel, and staurosporine was unable to reverse bistratene A-induced [3H]thymidine uptake inhibition, unlike TPA. Bistratene A did not induce PKC translocation or downregulation, activate or inhibit A549 and MCF-7 cell cytosolic PKC or compete for phorbol ester receptors. Western blot analysis and hydroxylapatite chromatography identified PKC α, ε and ζ in these cells. Bistratene A was unable to activate any of these isoforms. Therefore the agent does not exert its antiproliferative effects by modulation of PKC activity. The abilities of bryostatin 1 and TPA (10nM-1μM) to induce PKC isoform translocation and downregulation were compared with antiproliferative effects. Both agents induced dose-dependent downregulation and translocation of PKC α and ε to particulate and nuclear cell fractions. PKC ζ was translocated to the particulate fraction by both agents in MCF-7 cells. The similarity of PKC isoform redistribution by these agents did not explain their divergent effects on cell growth, and the role of nuclear translocation of PKC in cytostasis was not confirmed by these studies. Alternative factors governing the characteristics of growth inhibition induced by these agents are discussed.

The functional effects of ceramide on the monocyte CD36 scavenger receptor and NADPH oxidase

Atherosclerosis is the principal cause of death in the United States, Europe and much of Asia. During the last decade, inflammation has been suggested to play a key role in the development of atherosclerosis. Reactive oxygen species (ROS) released during inflammation additionally oxidize LDL, which is subsequently taken up in an unregulated way through scavenger receptors on macrophages to form foam cells, the hallmark of atherosclerotic lesions. Previous work has shown that the lipid ceramide, which is found in aggregated LDL and in atherosclerotic plaques, decreases intracellular peroxide most likely through reducing NADPH oxidase activity. Ceramide is an important component of membrane microdomains called lipid rafts which are important for membrane protein function. Endogenous ceramide enhances lipid raft f'ormation and alters theirs composition. NADPH oxidase membrane subunits cytochrome b558 (which includes gp91) strongly associates with lipid rafts Therefore present study investigated whether short chain ceramides reduce NADPH oxidase in U937 monocytes by disrurting the membrane component of NADPH oxidase. Results showed that C2 ceramide alters the distribution of raft marker, flottillin and the raft environment. NADPH oxidase membrane component gp9J phox and cytosolic component p47 phox were identified in rafts. C2 ceramide reduces both gp91 and p47 phox in rafts, which leads to the decrease of peroxide production by NADPH oxidase. Ceramide is also an important second messenger involved in many different signaling pathways associated with atherogenesis from the activation of sphingomyelinase (SMase). It has been reported that SMase enhances LDL receptor mediated LDL endocytosis. However, no study has been done to investigate the effect of ceramide on scavenger receptors such as CD36 and oxidized LDL (OxLDL) uptake. CD36 is the major recertor far OxLDL. Reduced CD36 expression results in less foam cell formation and less atherosclerotic lesion without disrupting the clearance of OxLDL from plasma. This thesis shows that ceramides significantly reduce CD36 surface expression on U937 monocytes, macrophages and human primary monocytes. This effect is seen using both synthetic short chain ceramide and SMase catalysed long chain ceramide treatment. To investigate whether the effect of ceramide on CD36 is functional, OxLOL uptake was measured in ceramide treated cells. Ceramide reduces the uptake of OxLOL by both U937 monocytes and PMA-differentiated macrophages. The mechanism of ceramide reduction of CD36 expression was studied by measuring the surface antigen using flow cytometry and fluorescence microscopy, whole cellular CD36 expression and shedding of C036 by Western blotting of cell lysates and cell culture supernatants and mRNA level of CD36 using RT-PCR. Ceramide reduces shedding of CD36, activates mRNA expression of CD36 and induces intracellular CD36 accumulation probably through retaining the receptor inside cells. In summary, ceramides modulate several of the processes involved in LOL oxidation and uptake by CD36 receptors on monocytes/macrophages in a way which may protect against atherosclerosis.

The neurotoxic effects of enzyme inhibitors at the neuromuscular junction

Current knowledge of the long-term, low dose effects of carbamate (CB) anti-cholinesterases on skeletal muscle or on the metabolism and regulation of the molecular forms of acetylcholinesterase (AChE) is limited. This is largely due to the reversible nature of these inhibitors and the subtle effects they induce which has generally made their study difficult and preliminary investigations were conducted to determine suitable study methods. A sequential extraction technique was used to rapidly analyse AChE molecular form activity at the mouse neuromuscular junction and also in peripheral parts of muscle fibres. AChE in the synaptic cleft involved in the termination of cholinergic transmission was successfully assessed by the assay method and by an alternative method using a correlation equation which represented the relationship between synaptic AChE and the prolongation of extra-cellular miniature endplate potentials. It was found that inhibition after in vivo Carbamate (CB) dosing could not be maintained during tissue analysis because CB-inhibited enzyme complexes decarbamoylated vary rapidly and could not be prevented even when maintained on ice. The methods employed did not therefore give a measure of inhibition but presented a profile of metabolic responses to continual, low dose CB treatment. Repetitive and continual infusion with low doses of the CBs: pyridostigmine and physostigmine induced a variety of effects on mouse skeletal muscle. Both compounds induced a mild myopathy in the mouse diaphragm during continual infusion which was characterised by endplate deformation without necrosis; such deformation persisted on termination of treatment but had recovered slightly 14 days later. Endplate and non-endplate AChE molecular forms displayed selective responses to CB treatment. During treatment endplate AChE was reduced whereas non-endplate AChE was largely unaffected, and after treatment, endplate AChE recovered, whereas non-endplate AChE was up-regulated. The mechanisms by which these responses become manifest are unclear but may be due to CB-induced effects on nerve-mediated muscle activity, neurotrophic factors or morphological and physiological changes which arise at the neuromuscular junction. It was concluded that, as well as inhibiting AChE, CBs also influence the metabolism and regulation of the enzyme and induce persistent endplate deformation; possible detrimental effects of long-term, low-dose determination requires further investigation.

In vivo effects of tailored laminin-332 α3 conjugated scaffolds enhances wound healing:a histomorphometric analysis

Surface modification techniques have been used to develop biomimetic scaffolds by incorporating cell adhesion peptides. In our previous work, we have shown the tethering of laminin-332 α3 chain to type I collagen scaffold using microbial transglutaminase (mTGase), promotes cell adhesion, migration, and proliferation. In this study, we evaluated the wound healing properties of tailored laminin-332 α3 chain (peptide A: PPFLMLLKGSTR) tethered to a type I collagen scaffold using mTGase by incorporating transglutaminase substrate peptide sequences containing either glutamine (peptide B: PPFLMLLKGSTREAQQIVM) or lysine (peptide C: PPFLMLLKGSTRKKKKG) in rat full-thickness wound model at two different time points (7 and 21 days). Histological evaluations were assessed for wound closure, epithelialization, angiogenesis, inflammatory, fibroblastic cellular infiltrations, and quantified using stereological methods (p < 0.05). Peptide A and B tethered to collagen scaffold using mTGase stimulated neovascularization, decreased the inflammatory cell infiltration and prominently enhanced the fibroblast proliferation which significantly accelerated the wound healing process. We conclude that surface modification by incorporating motif of laminin-332 α3 chain (peptide A: PPFLMLLK GSTR) domain and transglutaminase substrate to the laminin-332 α3 chain (peptide B: PPFLMLLKGSTREAQQIVM) using mTGase may be a potential candidate for tissue engineering applications and skin regeneration. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 101A:2788-2795, 2013. Copyright © 2013 Wiley Periodicals, Inc., a Wiley Company.

Effects of lithium and valproic acid on gene expression and phenotypic markers in an NT2 neurosphere model of neural development

Mood stabilising drugs such as lithium (LiCl) and valproic acid (VPA) are the first line agents for treating conditions such as Bipolar disorder and Epilepsy. However, these drugs have potential developmental effects that are not fully understood. This study explores the use of a simple human neurosphere-based in vitro model to characterise the pharmacological and toxicological effects of LiCl and VPA using gene expression changes linked to phenotypic alterations in cells. Treatment with VPA and LiCl resulted in the differential expression of 331 and 164 genes respectively. In the subset of VPA targeted genes, 114 were downregulated whilst 217 genes were upregulated. In the subset of LiCl targeted genes, 73 were downregulated and 91 were upregulated. Gene ontology (GO) term enrichment analysis was used to highlight the most relevant GO terms associated with a given gene list following toxin exposure. In addition, in order to phenotypically anchor the gene expression data, changes in the heterogeneity of cell subtype populations and cell cycle phase were monitored using flow cytometry. Whilst LiCl exposure did not significantly alter the proportion of cells expressing markers for stem cells/undifferentiated cells (Oct4, SSEA4), neurons (Neurofilament M), astrocytes (GFAP) or cell cycle phase, the drug caused a 1.4-fold increase in total cell number. In contrast, exposure to VPA resulted in significant upregulation of Oct4, SSEA, Neurofilament M and GFAP with significant decreases in both G2/M phase cells and cell number. This neurosphere model might provide the basis of a human-based cellular approach for the regulatory exploration of developmental impact of potential toxic chemicals.

Effects of metformin on BRIN-BD11 beta-cell insulin secretory desensitization induced by prolonged exposure to sulphonylureas

Aims: Prolonged exposure of pancreatic beta-cells in vitro to the sulphonylureas tolbutamide and glibenclamide induces subsequent desensitization of insulinotropic pathways. Clinically, the insulin-sensitizing biguanide drug metformin is often administered alongside sulphonylurea as antidiabetic therapy. The present study examines the functional effects of metformin (200 µM) on tolbutamide- and glibenclamide-induced desensitisation. Methods: Acute and prolonged (18 h) effects of exposure to tolbutamide and glibenclamide alone, or in the presence of metformin, were examined in insulin-secreting BRIN-BD11 cells. Results: In acute 20 min incubations at 1.1 mM glucose, metformin increased (1.2-1.7-fold; p <0.001) the insulin-releasing actions of tolbutamide and glibenclamide. At 16.7 mM glucose, metformin significantly enhanced glibenclamide-induced insulin release at all concentrations (50-400 µM) examined, but tolbutamide-stimulated insulin secretion was only augmented at higher concentrations (300-400 µM). Exposure for 18 h to 100 µM tolbutamide or glibenclamide significantly impaired insulin release in response to glucose and a broad range of insulin secretagogues. Concomitant culture with metformin (200 µM) prevented or partially reversed many of the adverse effects on K channel dependent and independent insulinotropic pathways. Beneficial effects of metformin were also observed in cells exposed to glibenclamide for 18 h with significant improvements in the insulin secretory responsiveness to alanine, GLP-1 and sulphonylureas. The decrease of viable cell numbers observed with glibenclamide was reversed by co-culture with metformin, but cellular insulin content was depressed. Conclusions: The results suggest that metformin can prevent the aspects of sulphonylurea-induced beta-cell desensitization. © 2010 Blackwell Publishing Ltd.

Disparate effects of non-steroidal anti-inflammatory drugs on apoptosis in guinea-pig gastric mucous cells: inhibition of basal apoptosis by diclofenac

Non-steroidal anti-inflammatory drugs (NSAIDs) induce apoptosis in gastrointestinal cancer cell lines. Similar actions on normal gastric epithelial cells could contribute to NSAID gastropathy. The present work therefore compared the actions of diclofenac, ibuprofen, indomethacin, and the cyclo-oxygenase-2 selective inhibitor, NS-398, on a primary culture of guinea-pig gastric mucous epithelial cells. Cell number was assessed by staining with crystal violet. Apoptotic activity was determined by condensation and fragmentation of nuclei and by assay of caspase-3-like activity. Necrosis was evaluated from release of cellular enzymes. Ibuprofen (250 μM for 24 h) promoted cell loss, and apoptosis, under both basal conditions and when apoptosis was increased by 25 μM N-Hexanoyl-D-sphingosine (C6-ceramide). Diclofenac (250 μM for 24 h) reduced the proportion of apoptotic nuclei from 5.2 to 2.1%, and caused inhibition of caspase-3-like activity, without causing necrosis under basal conditions. No such reduction in apoptotic activity was evident in the presence of 25 μM C6-ceramide. The inhibitory effect of diclofenac on basal caspase-3-like activity was also exhibited by the structurally similar mefenamic and flufenamic acids (1–250 μM), but not by niflumic acid. Inhibition of superoxide production by the cells increased caspase-3-like activity, but the inhibitory action of diclofenac on caspase activity remained. Diclofenac did not affect superoxide production. Diclofenac inhibited caspase-3-like activity in cell homogenates and also inhibited human recombinant caspase-3. In conclusion, NSAIDs vary in their effect on apoptotic activity in a primary culture of guinea-pig gastric mucous epithelial cells, and the inhibitory effect of diclofenac on basal apoptosis could involve an action on caspase activity.

Differential effects of chlorinated and oxidized phospholipids in vascular tissue:implications for neointima formation

The presence of inflammatory cells and MPO (myeloperoxidase) in the arterial wall after vascular injury could increase neointima formation by modification of phospholipids. The present study investigates how these phospholipids, in particular oxidized and chlorinated species, are altered within injured vessels and how they affect VSMC (vascular smooth muscle cell) remodelling processes. Vascular injury was induced in C57BL/6 mice and high fat-fed ApoE-/- (apolipoprotein E) mice by wire denudation and ligation of the left carotid artery (LCA). Neointimal and medial composition was assessed using immunohistochemistry and ESI-MS. Primary rabbit aortic SMCs (smooth muscle cells) were utilized to examine the effects of modified lipids on VSMC proliferation, viability and migration at a cellular level. Neointimal area, measured as intima-to-media ratio, was significantly larger in wire-injured ApoE-/- mice (3.62±0.49 compared with 0.83±0.25 in C57BL/6 mice, n=3) and there was increased oxidized low-density lipoprotein (oxLDL) infiltration and elevated plasma MPO levels. Relative increases in lysophosphatidylcholines and unsaturated phosphatidylcholines (PCs) were also observed in wire-injured ApoE-/- carotid arteries. Chlorinated lipids had no effect on VSMC proliferation, viability or migration whereas chronic incubation with oxidized phospholipids stimulated proliferation in the presence of fetal calf serum [154.8±14.2% of viable cells at 1 μM PGPC (1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine) compared with control, n=6]. In conclusion, ApoE-/- mice with an inflammatory phenotype develop more neointima in wire-injured arteries and accumulation of oxidized lipids in the vessel wall may propagate this effect.

Effects of lipoic acid and dihydrolipoic acid on total erythrocytic thiols under conditions of restricted glucose in vitro

The effects of lipoic acid and dihydrolipoic acid were explored on total thiol maintenance in diabetic and non-diabetic human erythrocytes in vitro over 22 hr in a 37°C incubation system with no added glucose. Over 18-22.5 hr after treatment in both non-diabetic and diabetic cells, lipoic acid (1 mM) was associated with greater loss of cellular thiols than dihydrolipoic acid (1 mM), compared to respective control values. At 0.1 mM, in non-diabetic cells, although lipoic acid-treated cells' thiol levels were significantly lower than control, there was no significant difference between dihydrolipoic acid-treated cells and control cells regarding thiol levels. In addition, at 0.1 mM, dihydrolipoic acid-treated diabetic cells showed a reduction in thiol levels compared to control. At 0.01 mM, lipoic acid-treated cells had significantly lower measured thiol levels compared with diabetic cells exposed to dihydrolipoic acid, whereas in non-diabetic cells, dihydrolipoic acid-treated erythrocytic thiol levels were significantly greater than those treated with lipoic acid, although there were no other significant differences between the groups. At 22.5 hr, control values of methaemoglobin rose to 6.4 ± 1.1% in diabetic cells and 3.6 ± 2.1% in non-diabetic cells. Lipoic acid (1 mM) showed greater methaemoglobin formation in diabetic rather than non-diabetic cells (13.6 ± 1.5% versus 11.6 ± 1.5%), whereas dihydrolipoic acid-treated diabetic and non-diabetic cells were less potent in methaemoglobin generation (8.5 ± 2.4% and 8.4 ± 1.4%, respectively). These studies suggest that in certain circumstances such as hypoglycaemia, lipoic acid administration may actually be detrimental to cellular oxidant protection status. © 2006 The Authors.