Marta Rybicka

Enhanced matrix-degrading proteolytic activity within the thin thrombus-covered wall of human abdominal aortic aneurysms

OBJECTIVE The maintenance of an arterial elastins integrity is essential in the prevention of abdominal aortic aneurysm (AAA) development. So far, the effect of intraluminal thrombus (ILT) thickness on the elastolytic activity within the AAA wall has not been studied. In the present study the hypothesis that thin thrombus is associated with enhanced proteolytic activity within human AAA wall was investigated. METHODS The specimens for analysis, from both thin (< or = 10 mm) thrombus-covered and thick (> or = 25 mm) thrombus-covered wall, had been taken from 40 patients undergoing elective repair of AAA. We evaluated neutrophil elastase activity with the enzymatic assay. Concentrations of active matrix metalloproteinase-9 (MMP-9), total matrix metalloproteinase-8 (MMP-8), and tissue inhibitor of matrix metalloproteinases-1 (TIMP-1) were measured by ELISA. Biochemical parameters were compared with the Wilcoxon signed-rank test. RESULTS Statistical analysis showed that the activity of elastase (P<0.0001) as well as concentrations of active MMP-9 (P=0.001), total MMP-8 (P<0.0001) and active MMP-9/total TIMP-1 ratio (P=0.002) were significantly higher in the thin thrombus-covered wall. Furthermore the TIMP-1 was found to have a lower concentration in the thin thrombus-covered in comparison with the thick thrombus-covered wall (P=0.003). There was a significant positive correlation between measurements in AAA wall sites with thin and thick thrombus for elastase, TIMP-1, MMP-9/TIMP-1 ratio, and a borderline correlation was observed for MMP-8. Active MMP-9 concentration did not correlate between sites. CONCLUSION The current study demonstrates the differentiation of protease activity within the same AAA wall and its enhancement within the thin thrombus-covered aneurysm wall.

Increased circulating endothelial progenitor cells in patients with haemorrhagic and ischaemic stroke: The role of Endothelin-1

Ischaemic stroke induces endothelial progenitor cell (EPC) mobilisation from bone marrow into peripheral blood. Circulating EPCs play an important role in post-injury regeneration of vasculature, whereas endothelial cells (ECs) have been shown to reflect endothelial damage and may be responsible for increased Endothelin-1 (ET-1) expression. We investigated herein the association between numbers of circulating ECs and EPCs, the levels of soluble factors regulating their migration and function, and the clinical outcome in patients with haemorrhagic (HS) or ischaemic stroke (IS). Sixteen patients with HS and eighteen with IS were assessed during the first 24h, day 3, and day 7 after stroke and compared them with twenty-three control subjects. We found elevated EPC and EC concentrations using flow cytometry and increase in VEGF, SDF-1, HGF, and ET-1 plasma levels by ELISA in the HS patients, while ET-1 mRNA expression in peripheral blood cells was elevated in the IS patients. Significant correlations were observed between EPCs or ECs and Big ET-1 protein or mRNA levels in HS but not in the IS patients. We suggest that ET-1 may play a role in pathophysiology of stroke and subsequent EPC mobilisation; however, further studies aimed at the precise elucidation of this issue are required.

Comparative effects of conjugated linoleic acid (CLA) and linoleic acid (LA) on the oxidoreduction status in THP-1 macrophages.

The aim of this study was to investigate the effect of conjugated linoleic acids (CLAs) on macrophage reactive oxygen species synthesis and the activity and expression of antioxidant enzymes, catalase (Cat), glutathione peroxidase (GPx), and superoxide dismutase (SOD). The macrophages were obtained from the THP-1 monocytic cell line. Cells were incubated with the addition of cis-9,trans-11 CLA or trans-10,cis-12 CLA or linoleic acid. Reactive oxygen species (ROS) formation was estimated by flow cytometry. Enzymes activity was measured spectrophotometrically. The antioxidant enzyme mRNA expression was estimated by real-time reverse transcriptase polymerase chain reaction (RT-PCR). Statistical analysis was based on nonparametric statistical tests [Friedman analysis of variation (ANOVA) and Wilcoxon signed-rank test]. cis-9,trans-11 CLA significantly increased the activity of Cat, while trans-10,cis-12 CLA notably influenced GPx activity. Both isomers significantly decreased mRNA expression for Cat. Only trans-10,cis-12 significantly influenced mRNA for SOD-2 expression. The CLAs activate processes of the ROS formation in macrophages. Adverse metabolic effects of each isomer action were observed.

The effect of perinatal lead exposure on dopamine receptor D2 expression in morphine dependent rats.

The aim of this study was to investigate the behavioral and molecular effects of pre- and postnatal lead (Pb) exposure on the expression of morphine withdrawal and tolerance in adult rats. Rats were orally treated with 0.1% (1000ppm) lead acetate from conception, through gestation, up to postnatal day (PND) 28. Subsequently, behavioral experiments were conducted on adult (PND 60) male rats. To assess behavioral effects of morphine dependence in Pb-exposed rats two experimental models were used: naloxone-precipitated withdrawal signs and the assessment of morphine tolerance to antinociceptive effect in the tail-immersion test. Morphine withdrawal and tolerance were more expressed in Pb-exposed morphine administered rats than in morphine administered rats. In the case of morphine withdrawal signs the analysis of protein (Western blotting) and mRNA (RT PCR) expression revealed significantly higher dopamine D2 receptor (D2R) expression in prefrontal cortex, but not in striatum and hippocampus, in Pb-exposed morphine administered rats than in morphine administered rats. Differently, in the case of morphine tolerance the significant upregulation of D2R protein and mRNA expression in hippocampus, but not in prefrontal cortex or striatum, was demonstrated in Pb-exposed and morphine administered rats in comparison with morphine administered. These findings suggest that in morphine withdrawal and tolerant rats the perinatal Pb-exposure can affect D2R expression in brain region-specific manner. Immunohistochemical assessment of D2R expression in hippocampus showed translocation of D2R from membrane-cytoplasm in control rats to nucleus in morphine administered rats. Perinatal Pb-exposure did not induce the changes in the localization of D2R irrespective of morphine effect.

Lead enhances fluoride influence on apoptotic processes in the HepG2 liver cell line

Chronic long-term exposure to high levels of fluoride leads to fluorosis, manifested by skeletal fluorosis and damage to internal organs, including kidneys, liver, parathyroid glands, and brain. Excess fluoride can also cause DNA damage, trigger apoptosis, and change cell cycle. The effect of fluoride may be exacerbated by lead (Pb), a potent inhibitor of many enzymes and a factor causing apoptosis, still present in the environment in excessive amounts. Therefore, in this study, we investigated the effects of sodium fluoride (NaF) and/or lead acetate (PbAc) on development of apoptosis, cell vitality, and proliferation in the liver cell line HepG2. We examined hepatocytes from the liver cell line HepG2, incubated for 48 h with NaF, PbAc, and their mixture (NaF + PbAc), and used for measuring apoptosis, index of proliferation, and vitality of cells. Incubation of the hepatocytes with NaF or PbAc increased apoptosis, more when fluoride and Pb were used simultaneously. Vitality of the cells depended on the compound used and its concentration. Proliferation slightly increased and then decreased in a high fluoride environment; it decreased significantly after addition of Pb in a dose-dependent manner. When used together, fluoride inhibited the decreasing effect of Pb on cell proliferation.

The character of haemostatic disorders and level of protein S-100 in acute ischaemic stroke can affect survival in the first week of follow-up: a pilot study.

Disorders of haemostasis which result in ischaemic stroke usually appear as thromboembolism in peripheral veins and the pulmonary circulation, and to a lesser extent as coagulopathy. The S-100 protein, a marker of stroke, correlates positively with the neurological deficit National Institutes of Health Stroke Scale (NIHSS). We adopted the hypothesis that early death of patients with acute ischaemic stroke can be explained by changes in blood coagulation and fibrinolysis. The study included 84 patients hospitalized with acute ischaemic stroke. Three groups were created: I (death between 1 and 2 days), II (death between 5 and 7 days) and III (with no deaths in hospital). We measured levels of fibrinogen, antithrombin, D-dimers, plasmin–antiplasmin complexes, plasminogen and clotting times (prothrombin time and activated partial thromboplastin time), platelet number, euglobulin clot lysis time (ECLTindex) and S-100 protein, C-reactive protein and white blood cells (WBCs). Group I had lower concentrations of fibrinogen compared to groups II (3.13 vs. 4.18, P < 0.01) and III (3.13 vs. 3.77, P < 0.02) and higher levels of D-dimers (3643 vs. 2278, P < 0.05), higher concentrations of plasmin–antiplasmin complexes (1410 vs. 882, P = 0.03) and a lower ECLTindex (152 vs. 219, P < 0.02) when compared with group III. Group I also had higher concentrations of protein S-100 (2.09 vs. 0.61, P < 0.001), higher NIHSS (18.0 vs. 13.2, P = 0.073) and number of WBC (14.1 vs. 11.1, P < 0.02) than in group III. The observed abnormalities in haemostasis, either found systemically or locally as cerebral microvascular thrombosis, may be factors potentially associated with death of patients with the shortest survival time.