fibrin and pimonidazole

Cotreatment of rats with nontoxic doses of ranitidine (RAN) and lipopolysaccharide (LPS) causes liver injury, and this drug-inflammation interaction might be a model for idiosyncratic adverse drug responses in humans. Both polymorphonuclear neutrophils (PMNs) and the hemostatic system have been shown to be important in the injury. We tested the hypothesis that PMNs cause liver injury by interacting with the hemostatic system and producing subsequent hypoxia. In rats cotreated with LPS/RAN, PMN depletion by anti-PMN serum reduced fibrin deposition and hypoxia in the liver. PMN depletion also reduced the plasma concentration of active plasminogen activator inhibitor-1 (PAI-1), a major down-regulator of the fibrinolytic system. This suggests that PMNs promote fibrin deposition by increasing PAI-1 concentration. PMNs were activated in the livers of LPS/RAN-cotreated rats as evidenced by increased staining for hypochlorous acid-modified proteins generated by the myeloperoxidase-hydrogen peroxide-chloride system of activated phagocytes. Antiserum against the PMN adhesion molecule CD18 protected against LPS/RAN-induced liver injury. Because CD18 is important for PMN transmigration and activation, these results suggest that PMN activation is required for the liver injury. Furthermore, anti-CD18 serum reduced biomarkers of hemostasis and hypoxia, suggesting the necessity for PMN activation in the interaction between PMNs and the hemostatic system/hypoxia. Liver injury, liver fibrin, and plasma PAI-1 concentration were also reduced by eglin C, an inhibitor of proteases released by activated PMNs. In summary, PMNs are activated in LPS/RAN-cotreated rats and participate in the liver injury in part by contributing to hemostasis and hypoxia.

Topics: Alanine Transaminase; Animals; Antithrombins; Cathepsin G; Cathepsins; CD18 Antigens; Fibrin; Fibrinogen; Hemostasis; Hypochlorous Acid; Immune Sera; Leukocyte Count; Lipopolysaccharides; Liver; Male; Models, Biological; Neutrophils; Nitroimidazoles; Pancreatic Elastase; Plasminogen Activator Inhibitor 1; Proteins; Ranitidine; Rats; Rats, Sprague-Dawley; Serine Endopeptidases; Serine Proteinase Inhibitors; Thrombin

The distribution of hypoxic cells in an in vivo tissue engineering chamber was investigated up to 28 days post-implantation.. Arteriovenous loops were constructed and placed into bi-valved polycarbonate chambers containing 2 x 10(6) rat fibroblasts in basement membrane gel (BM gel). Chambers were inserted subcutaneously in the groin of male rats and harvested at 3 (n = 6), 7 (n = 6), 14 (n = 4) or 28 (n = 4) days. Ninety minutes before harvest, pimonidazole (60 mg/kg) was injected intraperitoneally. Chamber tissue was removed, immersion fixed, paraffin embedded, sectioned and stained immunohistochemically using hypoxyprobe-1 Mab that detects reduced pimonidazole adducts forming in cells, where pO2 < 10 mmHg.. At 3 days a fibrin clot/BM gel framework filled the chamber. Seeded fibroblasts had largely died. The majority of 3 day chambers did not demonstrate tissue growth from the AV loop nor was pimonidazole binding present in these chambers. In one chamber in which tissue growth had occurred strong pimonidazole binding was evident within the new tissue. In four out of six 7 day chambers a broader proliferative zone existed extending up to 0.4 mm (approximately) from the AV loop endothelium which demonstrated intense pimonidazole binding. The two remaining 7 day chambers displayed even greater tissue growth (leading edge > 0.7 mm from the AV loop endothelium), but very weak or no pimonidazole binding. At 14 and 28 days the fibrin/BM gel matrix was replaced by mature vascularised connective tissue that did not bind pimonidazole.. Employing a tissue engineering chamber, new tissue growth extending up to 0.4 mm from the AV loop endothelium (chambers < or = 7 days) demonstrated intense pimonidazole binding and, therefore, hypoxia. Tissue growth greater than 0.5 mm from the AV loop endothelium (7-28 days chambers) did not exhibit pimonidazole binding due to a significant increase in the number of new blood vessels and was, therefore, adequately oxygenated.

Topics: Animals; Arteriovenous Shunt, Surgical; Biomarkers; Cell Division; Cell Hypoxia; Cell Movement; Cells, Cultured; Diffusion Chambers, Culture; Endothelium, Vascular; Fibrin; Fibroblasts; Gels; Male; Nitroimidazoles; Rats; Rats, Sprague-Dawley; Tissue Engineering