Huveyda Basaga

Pathological aspects of lipid peroxidation.

Abstract Lipid peroxidation (LPO) product accumulation in human tissues is a major cause of tissular and cellular dysfunction that plays a major role in ageing and most age-related and oxidative stress-related diseases. The current evidence for the implication of LPO in pathological processes is discussed in this review. New data and literature review are provided evaluating the role of LPO in the pathophysiology of ageing and classically oxidative stress-linked diseases, such as neurodegenerative diseases, diabetes and atherosclerosis (the main cause of cardiovascular complications). Striking evidences implicating LPO in foetal vascular dysfunction occurring in pre-eclampsia, in renal and liver diseases, as well as their role as cause and consequence to cancer development are addressed.

Lipid oxidation products in cell signaling

The recent research on the impact that oxidative changes of biolipids could have in pathophysiology serves to explain how free radical-driven reactions not only are considered as mere toxicologic events, but also modulators of cell activity and function. Oxidatively modified low-density lipoproteins are known to affect various cellular processes by modulating various molecular pathways and signaling nuclear transcription. Among the lipid oxidation products detectable in ox-LDLs, and also in the atherosclerotic plaques, 4-hydroxynonenal has been widely investigated. This aldehyde was shown to upregulate AP-1 transcription factor, signaling through the MAP kinase pathway, with eventual nuclear localization and induction of a series of genes. Further, oxidation products of cholesterol and cholesterol esters, in ox-LDL are of similar interest, especially in relation to the pathogenesis of fibrosclerotic lesions of the arterial wall.

Tumor suppressor genes and ROS: complex networks of interactions

Tumor suppressor genes regulate diverse cellular activities including DNA damage repair, cell cycle arrest, mitogenic signaling, cell differentiation, migration, and programmed cell death. In this review the tumor suppressor genes p53, FoxO, retinoblastoma (RB), p21, p16, and breast cancer susceptibility genes 1 and 2 (BRCA1 and BRCA2) and their roles in oxidative stress are summarized with a focus on the links and interplay between their pathways and reactive oxygen species (ROS). The results of a number of studies have demonstrated an antioxidant role for tumor suppressor proteins, activating the expression of some well-known antioxidant genes in response to oxidative stress. On the other hand, recent studies have revealed a pro-oxidant role for p53 by which cellular ROS are increased by enhanced transcription of proapoptotic genes. A tightly regulated feedback loop between ROS and FoxO proteins, with ROS regulating FoxO activity through posttranslational modifications and protein interactions and FoxO controlling intracellular ROS levels, has been demonstrated. Furthermore, these studies have shown that FoxO transcription factors and p38 mitogen-activated protein kinases may interact with the RB pathway under stress conditions. In addition, cellular senescence studies established an unexpected role for ROS in inducing and maintaining senescence-induced tumor suppression that blocks cytokinesis to ensure senescent cells never divide again. p21 and p16 have been shown to act as tumor suppressor proteins and this function extends beyond cell cycle control and includes important roles in regulating oxidative stress. Consequently, these important interactions indicate a critical potential role for tumor suppressor genes in the cellular response against oxidative stress and emphasize links between ROS and tumor suppressor genes that might be therapeutic targets in oxidative damage-associated diseases.

Fibronectin-Tissue Transglutaminase Matrix Rescues RGD-impaired Cell Adhesion through Syndecan-4 and β1 Integrin Co-signaling

Heterotropic association of tissue transglutaminase (TG2) with extracellular matrix-associated fibronectin (FN) can restore the adhesion of fibroblasts when the integrin-mediated direct binding to FN is impaired using RGD-containing peptide. We demonstrate that the compensatory effect of the TG-FN complex in the presence of RGD-containing peptides is mediated by TG2 binding to the heparan sulfate chains of the syndecan-4 cell surface receptor. This binding mediates activation of protein kinase Cα (PKCα) and its subsequent interaction with β1 integrin since disruption of PKCα binding to β1 integrins with a cell-permeant competitive peptide inhibits cell adhesion and the associated actin stress fiber formation. Cell signaling by this process leads to the activation of focal adhesion kinase and ERK1/2 mitogen-activated protein kinases. Fibroblasts deficient in Raf-1 do not respond fully to the TG-FN complex unless either the full-length kinase competent Raf-1 or the kinase-inactive domain of Raf-1 is reintroduced, indicating the involvement of the Raf-1 protein in the signaling mechanism. We propose a model for a novel RGD-independent cell adhesion process that could be important during tissue injury and/or remodeling whereby TG-FN binding to syndecan-4 activates PKCα leading to its association with β1 integrin, reinforcement of actin-stress fiber organization, and MAPK pathway activation.

Up-regulation of the fibrogenic cytokine TGF-beta1 by oxysterols: a mechanistic link between cholesterol and atherosclerosis.

Deposition of blood cholesterol in the subendothelial space of major arteries is a main feature of the fibrotic plaque as well as the key event in the progression of atherosclerosis. However, the mechanisms by which cholesterol triggers and sustains the fibrotic degeneration of blood arteries remain undefined. The results reported here indicate that a biologically representative mixture of oxysterols, 27‐carbon products of cholesterol oxidation, rather than an individual oxysterol at the same concentration, exerts a strong profibrogenic effect when taken up by macrophages. Incubation of murine and human macrophages with such oxysterol mixture markedly promotes both expression and synthesis of one of the most potent proinflammatory and fibrogenic cytokines, transforming growth factor β1 (TGF‐β1). By contrast, no effect on TGF‐β1 expression and synthesis was found with unoxidized cholesterol. Macrophage uptake of cholesterol and conversion to foam cells is a hallmark of early atherogenesis, but it appears that cholesterol oxidation products, as well as other low‐density lipoprotein oxidation products, are required to generate a proper fibrogenic stimulus that is not fulfilled by cholesterol deposition alone.

Protein kinases as drug targets in cancer

Identification of the key roles of protein kinases in signaling pathways leading to development of cancer has caused pharmacological interest to concentrate extensively on targeted therapies as a more specific and effective way for blockade of cancer progression. This review will mainly focus on inhibitors targeting these key components of cellular signaling by employing a technology-based point of view with respect to ATP- and non-ATP-competitive small molecule inhibitors and monoclonal antibodies of selected protein kinases, particularly, mammalian target of rapamycin (mTOR), BCR-ABL, MEK, p38 MAPK, EGFR PDGFR, VEGFR, HER2 and Raf. Inhibitors of the heat shock protein Hsp90 are also included in a separate section, as this protein plays an essential role for the maturation/proper activation of cancer-related protein kinases. In the following review, the molecular details of the mode of action of these inhibitors as well as the emergence of drug resistance encountered in several cases are discussed in light of the structural, molecular and clinical studies conducted so far.

Studies on the antioxidant and free radical scavenging properties of IdB 1016 a new flavanolignan complex.

Silybin has been complexed in 1:1 ratio with phosphatidyl choline to give IdB 1016 in order to increase its bioavailability. The antioxidant and free radical scavenger action of this new form of silybin has been evaluated. One hour after the intragastric administration to rats of IdB 1016 (1.5 g/kg b.wt.) the concentration of silybin in the liver microsomes was estimated to be around 2.5 micrograms/mg protein corresponding to a final concentration in the microsomal suspension used of about 10 microM. At these levels IdB decreased by about 40% the lipid peroxidation induced in microsomes by NADPH, CCl4 and cumene hydroperoxide, probably by acting on lipid derived radicals. Spin trapping experiments showed, in fact, that the complexed form of silybin was able to scavenge lipid dienyl radicals generated in the microsomal membranes. In addition, IdB 1016 was also found to interact with free radical intermediates produced during the metabolic activation of carbon tetrachloride and methylhydrazine. These effects indicate IdB 1016 as a potentially protective agent against free radical-mediated toxic damage.

Free Radical Scavenging and Antioxidative Properties of ‘Silibin’ Complexes on Microsomal Lipid Peroxidation

The antioxidant properties of silibin complexes, the water‐soluble form silibin dihemisuccinate (SDH), and the lipid‐soluble form, silibin phosphatidylcholine complex known as IdB 1016, were evaluated by studying their abilities to react with the superoxide radical anion (O2.−), and the hydroxyl radical (OH.). In addition, their effect on pulmonary and hepatic microsomal lipid peroxidation had been investigated. Superoxide radicals were generated by the PMS‐NADH system and measured by their ability to reduce NBT. IC50 concentrations for the inhibition of the NBT reduction by SDH and IdB 1016 were found to be 25 μM and 316 μM respectively. Both silibin complexes had an inhibitory effect on xanthine oxidase activity. SDH reacted rapidly with OH. radicals at approximately diffusion controlled rate and the rate constant was found to be (K=8·2×109 M−1 s−1); it appeared to chelate Fe2+ in solution.

Increased TG2 Expression Can Result in Induction of Transforming Growth Factor β1, Causing Increased Synthesis and Deposition of Matrix Proteins, Which Can Be Regulated by Nitric Oxide

In fibrotic conditions increases in TG2 activity has been linked to an increase in the deposition of extracellular matrix proteins. Using TG2 transfected Swiss 3T3 fibroblasts expressing TG2 under the control of the tetracycline-regulated inducible promoter, we demonstrate that induction of TG2 not only stimulates an increase in collagen and fibronectin deposition but also an increase in the expression of these proteins. Increased TG2 expression in these fibroblasts led to NF-κB activation, resulting in the increased expression of transforming growth factor (TGF) β1. In addition, cells overexpressing TG2 demonstrated an increase in biologically active TGFβ1 in the extracellular environment. A specific site-directed inhibitor of TG abolished the NF-κB and TGFβ1 activation and the subsequent elevation in the synthesis and deposition of extracellular matrix proteins, confirming that this process depends on the induction of transglutaminase activity. Treatment of TG2-induced fibroblasts with nontoxic doses of nitric oxide donor S-nitroso-N-acetylpenicillamine resulted in decreased TG2 activity and apprehension of the inactive enzyme on the cell surface. This was paralleled by a reduction in activation of NF-κB and TGFβ1 production with a subsequent decrease in collagen expression and deposition. These findings support a role for NO in the regulation of TG2 function in the extracellular environment.

Bcl-2 protein family: implications in vascular apoptosis and atherosclerosis.

Apoptosis has been recognized as a central component in the pathogenesis of atherosclerosis, in addition to the other human pathologies such as cancer and diabetes. The pathophysiology of atherosclerosis is complex, involving both apoptosis and proliferation at different phases of its progression. Oxidative modification of lipids and inflammation differentially regulate the apoptotic and proliferative responses of vascular cells during progression of the atherosclerotic lesion. Bcl-2 proteins act as the major regulators of extrinsic and intrinsic apoptosis signalling pathways and more recently it has become evident that they mediate the apoptotic response of vascular cells in response to oxidation and inflammation either in a provocative or an inhibitory mode of action. Here we address Bcl-2 proteins as major therapeutic targets for the treatment of atherosclerosis and underscore the need for the novel preventive and therapeutic interventions against atherosclerosis, which should be designed in the light of molecular mechanisms regulating apoptosis of vascular cells in atherosclerotic lesions.