Kamelija Zarkovic

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.

Oxidized Low-Density Lipoproteins Trigger Endoplasmic Reticulum Stress in Vascular Cells Prevention by Oxygen-Regulated Protein 150 Expression

Oxidized low-density lipoproteins (oxLDLs) trigger various biological responses potentially involved in atherogenesis. Disturbing endoplasmic reticulum (ER) function results in ER stress and unfolded protein response, which tends to restore ER homeostasis but switches to apoptosis when ER stress is prolonged. We aimed to investigate whether ER stress is induced by oxLDLs and can be prevented by the ER-associated chaperone ORP150 (150-kDa oxygen-regulated protein). oxLDLs and the lipid oxidation products 7-ketocholesterol and 4-hydroxynonenal induce ER stress in human endothelial cells (HMEC-1), characterized by the activation of ER stress sensors (phosphorylation of Ire1&agr; and PERK, nuclear translocation of ATF6) and of their subsequent pathways (eukaryotic initiation factor 2&agr; phosphorylation, expression of XBP1/spliced XBP1, CHOP, and KDEL chaperones GRP78, GRP94, ORP150). ER stress was inhibited by the antioxidant N-acetylcysteine. In advanced atherosclerotic lesions, phospho-Ire1&agr;, KDEL, and ORP150 staining were localized in lipid-rich areas with 4-hydroxynonenal adducts and CD68-positive macrophagic cells. By comparison, staining for 4-hydroxynonenal, phospho-Ire1&agr;, KDEL, and ORP were faint and more diffuse in intimal hyperplasia. ER stress takes part in the apoptotic effect of oxLDLs, through the Ire1&agr;/c-Jun N-terminal kinase pathway, as assessed by the protective effect of specific small interfering RNAs and c-Jun N-terminal kinase inhibitor. Forced expression of the chaperone ORP150 reduced both oxLDL-induced ER stress and apoptosis. ER stress markers and ORP150 chaperone are expressed in areas containing oxLDLs in atherosclerotic lesions and are induced by oxLDLs and oxidized lipids in cultured cells. The forced expression of ORP150 highlights its new protective role against oxLDL-induced ER stress and subsequent apoptosis.

Advances in methods for the determination of biologically relevant lipid peroxidation products

Abstract Lipid peroxidation is recognized to be an important contributor to many chronic diseases, especially those of an inflammatory pathology. In addition to their value as markers of oxidative damage, lipid peroxidation products have also been shown to have a wide variety of biological and cell signalling effects. In view of this, accurate and sensitive methods for the measurement of lipid peroxidation products are essential. Although some assays have been described for many years, improvements in protocols are continually being reported and, with recent advances in instrumentation and technology, highly specialized and informative techniques are increasingly used. This article gives an overview of the most currently used methods and then addresses the recent advances in some specific approaches. The focus is on analysis of oxysterols, F2-isoprostanes and oxidized phospholipids by gas chromatography or liquid chromatography mass spectrometry techniques and immunoassays for the detection of 4-hydroxynonenal.

Pathophysiological relevance of aldehydic protein modifications.

There is growing body of evidence that oxidative stress, i.e. excess in production of reactive oxygen species, can lead to covalent modification of proteins with bioactive aldehydes that are mostly produced under lipid peroxidation of polyunsaturated fatty acids. Thus generated reactive aldehydes are considered as second messengers of free radicals because they react with major bioactive macromolecules, in particular with various humoral and cellular proteins changing their structure and functions. Therefore, the aldehydic-protein adducts, in particular those involving 4-hydroxy-2-nonenal, malondialdehyde and acrolein can be valuable biomarkers of numerous pathophysiological processes. The development of immunochemical methods is increasing the possibilities to study such non-enzymatic protein modifications, on the one hand, while on the other hand the increase of knowledge on bioactivities of the aldehydes and their protein adducts might lead to better prevention, diagnosis and treatments of pathophysiological processes associated with lipid peroxidation and oxidative stress in general. This article is part of a Special Issue entitled: Posttranslational Protein modifications in biology and Medicine.

A fish oil-rich diet reduces vascular oxidative stress in apoE–/– mice

Abstract Oxidative stress contributes to lipid peroxidation and decreases nitric oxide (NO) bioavailability in atherosclerosis. While long-chain (n-3) polyunsaturated fatty acids (PUFA) are easily oxidized in vitro, they improve endothelial function. Hence, this study postulates that long-chain (n-3) PUFA decrease atherogenic oxidative stress in vivo. To test this, apoE–/– mice were fed a corn oil- or a fish oil (FO)-rich diet for 8, 14 or 20 weeks and parameters related to NO and superoxide (O2.–) plus markers of lipid peroxidation and protein oxidative damage in the aortic root were evaluated. The FO-rich diet increased NO production and endothelial NO synthase (NOS) expression and lowered inducible NOS, p22phox expression and O2.–production after 14 and 20 weeks of diet. Protein lipoxidative damage (including 4-hydroxynonenal) was decreased after a long-term FO-diet. This supports the hypothesis that a FO-rich diet could counteract atherogenic oxidative stress, showing beneficial effects of long-chain (n-3) PUFA.

Mitochondrial alterations in aging rat brain: effective role of ( )-epigallo catechin gallate

Aging is a multi‐factorial process which involves deprivation in bodys metabolism. Brain mitochondria are prone to oxidative damage owing to their high metabolic rate. The decline in antioxidant system during aging augments the neuronal damage to mitochondrial components like antioxidant system, Krebs cycle enzymes and electron transport chain complexes. Since brain is an organ rich in fatty acids, lipid peroxidation products like hydroxynonenal are predominant. Those lipid peroxidation products conjugate with amino acids to form adducts which alter their structural and functional properties. Epigallo catechin gallate is a potent antioxidant which is rich in green tea extract. This study elucidated the antioxidant potential of epigallo catechin gallate to counteract the mitochondrial oxidative damage in brain. The study comprised of young (3–4 months old; 150 ± 20 g) and aged (above 24 months; 420 ± 20 g) male albino rats of Wistar strain in Groups I and II. Groups III and IV comprised of young and aged rats supplemented with epigallo catechin gallate (2 mg/kg body weight) for 30 days. Antioxidants, Krebs cycle enzymes and electron transport chain complexes were assayed in the mitochondrial fraction. Hydroxynonenal expression was carried out using immunohistochemical analysis. Epigallo catechin gallate supplementation decreased the expression of hydroxynonenal in aged brain, up‐regulated the antioxidant system and augmented the activities of Krebs cycle enzymes and electron transport chain complexes in aged brain mitochondria thus proving its antioxidant potential at the level of mitochondria.

Basic fibroblast growth factor (BFGF) immunoreactivity as a possible link between head injury and impaired bone fracture healing

Healing of fractures of long bones or large joints is often accelerated in patients with severe traumatic brain injury (TBI). However, in these patients an early fracture healing is accompanied by hypertrophic callus formation or heterotopic ossifications which might even result in an ankylosis of the affected joints. It seems that enhanced osteogenesis in patients suffering from TBI could be caused by some humoral factors, since the sera of these patients strongly promote the growth of osteoblast cells in vitro. However, humoral growth promoting factors which could perhaps induce enhanced osteogenesis are not yet identified. Hence, the aim of this study was to analyse if basic fibroblast growth factor (bFGF) could be related to the phenomenon of enhanced osteogenesis, since bFGF stimulates the growth of osteoblasts in vitro and could be found both in the brain and the bone tissue. For that purpose the values of bFGF immunoreactivity were determined in the sera of patients with TBI and bone fractures (n = 8) as well as in the sera of patients with either TBI alone (n = 10) or bone fractures alone (n = 7), during a period of three months after injury. Quantification of the bFGF immunoreactivity was done using the ELISA based on monoclonal antibodies raised against the recombinant human bFGF. The bFGF immunoreactivity values obtained were also compared with the values determined in the sera of normal, healthy persons (n = 9). In the group of patients with bone fractures alone only a transient increase of bFGF immunoreactivity (threefold above the normal values) was observed in the second week after injury. A similar increase of the values of bFGF immunoreactivity was also determined in the sera of patients with TBI only, but it lasted longer (from the 1st until the 7th to 8th week after injury). In the case of patients with TBI and bone fractures a specific pattern of post-traumatic dynamic change of the values of serum bFGF immunoreactivity was observed. Namely, the increase of bFGF immunoreactivity (up to seven-fold above the normal values) was determined even during the first week after injury. Afterwards, periods of high values of bFGF immunoreactivity observed during the 2nd, 4th and the 7-10th weeks after injury were interrupted by sudden decreases even to the normal values (during the 3rd and the 5-6th week after injury).(ABSTRACT TRUNCATED AT 400 WORDS)

Tissue distribution of lipid peroxidation product acrolein in human colon carcinogenesis

Lipid peroxidation product acrolein, well-known pollutant in tobacco and automotive smoke, accumulates in vivo bound to proteins. It suppresses p53 synthesis acting as potent carcinogenic factor for oral, respiratory and bladder carcinomas, while its possible association with colon carcinogenesis was not studied so far. We used genuine monoclonal antibody to evaluate immunohistochemical distribution of acrolein–protein adducts in 113 human colon tumours. The presence of acrolein–protein adducts was increasing with respect to colon carcinogenesis, from moderate appearance in tubular and villotubular low-grade adenomas to abundant and diffuse distribution in high-grade villotubular adenomas and Dukes A carcinomas. However, in advanced Dukes B and C carcinomas acrolein was hardly noticed, although, its protein adducts were found abundant in non-malignant colon epithelium of these patients. There was no relationship between p53 and acrolein distribution. According to these findings, acrolein seems to be lipid peroxidation product associated with transition from benign into malignant colon tumours.

Oxidative stress in small-for-gestational age (SGA) term newborns and their mothers.

This study used malondialdehyde (MDA) determination by HPLC and enzymatic assays for total serum peroxides and antioxidant capacity to evaluate oxidative stress in 47 healthy full-term small-for-gestational age (SGA) newborns vs 67 appropriate-for-gestational age (AGA) newborns. Blood samples were collected at delivery from umbilical cord artery and vein and from peripheral blood of the babies on the third day after birth. Blood samples of mothers were also collected and compared with blood of 29 normal non-pregnant women (NPW). Serum peroxide values were significantly higher in both groups of mothers than in NPW, decreasing towards the third day in AGA mothers, while persisting in SGA mothers. Antioxidant capacity of sera of both groups of mothers was lower than NPW. Both SGA mothers and babies had increased MDA at delivery, unlike AGA counterparts. MDA levels in umbilical vein were higher than in umbilical arteries, while immunohistochemistry revealed abundant presence of 4-hydroxynonenal (HNE)-protein adducts only in stroma of the SGA placenta. These results show that both mothers and babies are exposed to oxidative stress during and after delivery, which is more pronounced and persistent in the perinatal period of the SGA group, while lipid peroxidation in placenta could play a role in SGA pathophysiology.

Specific thermographic changes during Walker 256 carcinoma development: differential infrared imaging of tumour, inflammation and haematoma.

BACKGROUND Infrared imaging measures spatial variations in the skin temperature aiming to determine pathological processes; hence possible use of this non-invasive analytical method in cancer detection is emerging. METHODS Infrared thermal imaging was used to detect changes in rat skin surface temperature associated with experimental cancer development (Walker 256 carcinoma), inflammation (upon s.c. Sephadex injection) and haematoma (provoked by s.c. blood coagulate injection). Infrared camera with a geometric resolution of 76,800 pixels, spectral range of 8-14 microm and the minimal detectable temperature resolution of 0.07 degrees C with spatial resolution of 0.48 mm at measuring distance of 30 cm was used to obtain computerised thermal scans. Genuine ThermoWEB software developed for remote internet control as open source software was used. RESULTS The raise of peripheral temperature was observed after induction of local inflammation or haematoma. Opposite to that, transient decrease of the skin surface temperature was observed after tumour transplantation. Progressive growth of tumour was associated with the raise of the skin surface temperature from the 10th day after tumour inoculation, when the tumours developed supportive neoangiogenic blood supply, as verified by histology. CONCLUSION While the raise of peripheral temperature in advanced tumour was caused by neoangiogenesis, the reduction in skin surface temperature in an early period after tumour cell inoculation indicated a decay of transplanted tumour cells due to the immune response and the lack of blood supply. Thus, infrared thermal imaging may have considerable value in evaluation of the tumour development and discrimination of cancer from inflammation and haematoma.