Paolo Seminara

Attenuated Cerulein-Induced Pancreatitis in Nuclear Factor–κB–Deficient Mice

Nuclear factor (NF)-κB plays a central role in acute pancreatitis. We studied cerulein (CER)-induced pancreatitis in NF-κB knockout (KO) mice. NF-κB KO mice and normal control littermate wild-type (WT) mice were given four hyperstimulating doses of cerulein every hour to elicit secreatagogue-induced pancreatitis. Malonildialdehyde activity, glutathione levels, myeloperoxidase activity, TNF-α, and NF-κB binding activity and its inhibitory protein IκBα were studied in the pancreas. Furthermore, we measured plasma lipase and amylase and the histological damage. KO mice had reduced malonildialdehyde levels (WT + CER = 4.083 ± 0.95 μmol/g; KO + CER = 1.513 ± 0.63 μmol/g), decreased myeloperoxidase activity (WT + CER = 19.3 ± 2.39 mU/g; KO + CER = 10.21 ± 2.05 mU/g), increased glutathione levels (WT + CER 6.22 ± 2.46 μmol/g; KO + CER = 15. 516 ± 2.92 μmol/g), and reduced serum levels of amylase (WT + CER = 2519 ± 656.9 U/L; KO + CER = 916 ± 280.4 U/L) and lipase (WT + CER = 1420 ± 170 U/L; KO + CER = 861 ± 172. 3 U/L). KO mice showed reduced pancreatic NF-κB activation, decreased TNF-α tissue content, and reduced histologic alterations. Our data suggest that KO mice have an attenuated cerulein-induced pancreatitis and help to define the possible interaction between NF-κB activation and oxidative stress in this deleterious event.

Effect of recombinant adeno-associated virus vector-mediated vascular endothelial growth factor gene transfer on wound healing after burn injury.

ObjectiveThe purpose of this study was to investigate the effect of recombinant adeno-associated viral (rAAV) vector-mediated human vascular endothelial growth factor (VEGF165) transfer on experimental burn wounds. DesignRandomized experiment. SettingResearch laboratory. SubjectsC57BL/6 male mice weighing 25–30 g. InterventionsMice were immersed in 80°C water for 10 secs to achieve a partial-thickness scald burn. Animals were randomized to receive at two injection sites on the edge of the burn either 1011 copies of the rAAV-VEGF165 or the vector carrying the control and inert gene &bgr;-galactosidase (rAAV-LacZ). On day 14 the animals were killed. Burn areas were used for histologic examination, evaluation of VEGF expression (immunohistochemistry) and VEGF wound content (enzyme-linked immunosorbent assay), determination of wound nitrite, and measurement of messenger RNA (mRNA) for endothelial and inducible nitric oxide synthase (eNOS and iNOS). Measurements and Main ResultsrAAV-VEGF165 increased epithelial proliferation, angiogenesis, and maturation of the extracellular matrix. Furthermore, gene transfer enhanced VEGF expression, studied by immunohistochemistry, and the wound content of the mature protein (rAAV-LacZ, 11 ± 5 pg/wound; rAAV-VEGF165, 104 ± 7 pg/wound). Moreover, VEGF165 gene transfer increased wound content of nitrate. Finally, rAAV-VEGF165 administration enhanced the messenger RNA for eNOS (rAAV-VEGF165, 1.1 ± 0.2 relative amount of eNOS mRNA; rAAV-LacZ, 0.66 ± 0.3 relative amount of eNOS mRNA) and iNOS (rAAV-VEGF165, 0.8 ± 0.09 relative amount of iNOS mRNA; rAAV-LacZ, 0.45 ± 0.05 relative amount of iNOS mRNA). ConclusionOur study suggests that rAAV-VEGF gene transfer may be an effective therapeutic approach to improve clinical outcomes after thermal injury.

Modulation of IL-1 β gene expression by lipid peroxidation inhibition after kainic acid-induced rat brain injury

Brain injury was induced by intraperitoneal administration of kainic acid (KA, 10 mg/kg). Animals were randomized to receive either IRFI 042 (20 mg/kg i.p.), a lipid peroxidation inhibitor, or its vehicle (NaCl 0.9% DMSO 10% 1 ml/kg i.p.) 30 min before KA administration. A first set of animals was sacrificed 6 h after KA injection to measure malondialdehyde (MDA) content, glutathione-reduced (GSH) levels and the mRNA for interleukin-1beta (IL-1beta) in the cortex and in the hippocampus. A second set of animals was sacrificed 48 h after KA administration for histological analysis. All animals were observed for monitoring the behavioral sequelae and for evaluating latency of convulsions. Sham brain injury rats were used as controls. Intraperitoneal administration of IRFI 042 significantly decreased brain MDA (cortex: KA + vehicle = 0.285 +/- 0.04 nmol/mg protein; KA + IRFI 042 = 0.156 +/- 0.02 nmol/mg protein, P < 0.005; hippocampus: KA + vehicle = 0.350 +/- 0.03 nmol/mg protein; KA + IRFI 042 = 0.17 +/- 0.04 nmol/mg protein, P < 0.005), prevented the brain loss of GSH in both cortex (KA + vehicle = 7.81 +/- 1 micromol/g protein; KA + IRFI 042 = 12.1 +/- 1 micromol/g protein; P < 0.005) and hippocampus (KA + vehicle = 5 +/- 0.8 micromol/g protein; KA + IRFI 042 = 9.4 +/- 1.8 micromol/g protein; P < 0.005), reduced both brain IL-1beta mRNA expression and oedema, and increased latency of convulsions. Histological analysis showed a reduction of cell damage in IRFI 042-treated samples. The present data indicate that lipid peroxidation inhibition reduces IL-1beta gene expression and protects against kainic acid-induced brain damage.

Gene transfer of IκBα limits infarct size in a mouse model of myocardial ischemia-reperfusion injury

Nuclear factor-κB (NF-κB) plays a central role in myocardial ischemia-reperfusion (MI/R) injury. The inhibitory protein IκBα prevents its activation. We investigated the effects of adeno-associated viral vector-mediated IκBα gene transfer in MI/R injury. Male C57BL/6 mice were randomized to receive a recombinant adeno-associated virus (rAAV) encoding the gene for the NF-κB inhibitory protein IκBα (rAAV- IκBα) or the β-galactosidase gene (a control and inert gene; rAAV-LacZ), both at a dose of 1011 copies. Four weeks later anesthetized animals were subjected to total occlusion (45 minutes) of the left main coronary artery followed by 5 hours of reperfusion. MI/R produced a wide infarct size (IF/area-at-risk = 56 ± 8%; IF/left ventricle = 44 ± 5%) and tissue neutrophil infiltration, studied by means of elastase activity (area-at-risk = 2.5 ± 0.4 μg/gm tissue; infarct area = 2.9 ± 0.6 μg/gm tissue). Furthermore MI/R caused peak message for intercellular adhesion molecule-1 (ICAM-1) in the area-at-risk at 3 hours of reperfusion (1.2 ± 0.4 relative amount of cardiac ICAM-1 mRNA). NF-κB activation was evident at 0.5 hours of reperfusion and reached its maximum increase at 2 hours of reperfusion. rAAV-IκBα injection reduced infarct size (IF/area-at-risk = 19 ± 3%; IF/left ventricle = 10 ± 2%; p < 0.001), blocked NF-κB activation, diminished cardiac ICAM-1 expression (0.4 ± 0.02 relative amount of cardiac ICAM-1 mRNA; p < 0.001), and blunted leukocyte accumulation (area-at-risk = 0.6 ± 0.05 μg/gm tissue; infarct area = 0.4 ± 0.02 μg/gm tissue; p < 0.001). Our data indicate that rAAV-IκBα may be useful for MI/R gene therapy.

Lipid peroxidation inhibition reduces NF-κB activation and attenuates cerulein-induced pancreatitis

Increased lipid peroxidation, enhanced nuclear factor kappa-B (NF- s B) activation and augmented tumor necrosis factor- f (TNF- f ) production have been implicated in cerulein-induced pancreatitis. We investigated whether lipid peroxidation inhibition might reduce NF- s B activation and the inflammatory response in cerulein-induced pancreatitis. Male Sprague-Dawley rats of 230-250 g body weight received administration of cerulein (80 w g/kg s.c. for each of four injections at hourly intervals). A control group received four s.c. injections of 0.9% saline at hourly intervals. Animals were randomized to receive either raxofelast, an inhibitor of lipid peroxidation (20 mg/kg i.p. administered with the first cerulein injection) or its vehicle (1 ml/kg of a 10% DMSO/NaCl solution). All these rats were sacrificed 2 h after the last injection of either cerulein or its vehicle. Raxofelast administration (20 mg/kg i.p. with the first cerulein) significantly reduced malondialdehyde (MDA) levels, an index of lipid peroxidation (CER+DMSO=3.075 - 0.54 w mol/g; CER+raxofelast= 0.693 - 0.18 w mol/g; p <0.001 ), decreased myeloperoxidase (MPO) activity ( CER+DMSO=22.2 - 3.54 mU/g; CER+raxofelast=9.07 - 2.05 mU/g; p <0.01 ), increased glutathione levels (GSH) (CER+DMSO= 5.21 - 1.79 w mol/g; CER+raxofelast=15.71 - 2.14 w mol/g; p <0.001 ), and reduced acinar cell damage evaluated by means of histology and serum levels of both amylase ( CER+DMSO=4063 - 707.9 U/l; CER+raxofelast=1198 - 214.4 U/l; p <0.001 ), and lipase (CER+DMSO=1654 - 330 U/l; CER+raxofelast= 386 - 118.2 U/l; p <0.001 ), Furthermore, raxofelast reduced pancreatic NF- s B activation and the TNF- f mRNA levels and tissue content of mature protein in the pancreas. Indeed, lipid peroxidation inhibition might be considered a potential therapeutic approach to prevent the severe damage in acute pancreatitis.

Lipid peroxidation inhibition by raxofelast improves angiogenesis and wound healing in experimental burn wounds.

We investigated the effects of raxofelast, a lipid peroxidation inhibitor, in an experimental model of burn wounds. C57BL/6 male mice of 25-30 g were immersed in 80°C water for 10 seconds to achieve a partial-thickness scald burn. Animals received intraperitoneally either raxofelast (20 mg/kg/day for 14 days in 100 μL) or its vehicle alone (100 μL/day for 14 days). On day 14, burn areas were used for measuring conjugated dienes, reduced glutathione levels, histological damage, neoangiogenesis by immunohistochemistry and expression (Western blot) of the specific endothelial marker CD31 as well as quantification of microvessel density, VEGF wound content, endothelial and inducible nitric oxide synthase (eNOS and iNOS) expression and wound nitrite content. Raxofelast decreased tissue conjugated dienes (vehicle 6.1 ± 1.4 ΔABS/mg protein; raxofelast 3.7 ± 0.8 ΔABS/mg protein), prevented tissue glutathione consumption (vehicle 3.2 ± 0.9 μmol/g protein; raxofelast 6.7 ± 1.8 μmol/g protein), increased epithelial proliferation, extracellular matrix maturation, and augmented neoangiogenesis as suggested by the marked increase in microvessel density and by the robust expression of the specific endothelial marker CD31 (vehicle 9.4 ± 1.1 integrated intensity; raxofelast 14.8 ± 1.8 integrated intensity). Furthermore, raxofelast enhanced VEGF wound content (vehicle 1.4 ± 0.4 pg/mg protein; raxofelast 2.4 ± 0.6 pg/mg protein), caused a marked expression of eNOS (vehicle 16.1 ± 3 integrated intensity; raxofelast 26.2 ± 4 integrated intensity) and iNOS (vehicle 9.1 ± 1.8 integrated intensity; raxofelast 16.2 ± 3.5 integrated intensity) and increased wound nitrite content. Lipid peroxidation inhibition by raxofelast may be an effective therapeutic approach to improve clinical outcomes after thermal injury.

Protective Effects of Antioxidant Raxofelast in Alcohol-Induced Liver Disease in Mice

We investigated the effect of raxofelast on lipid peroxidation inhibition in mice exposed to chronic ethanol. Female C57BL/6 mice were fed a modified Lieber-DeCarli liquid ethanol (ETOH) or control diet (sham ETOH) for up to 14 days. Animals were assigned to receive either raxofelast (20 mg/kg/day i.p.) or its vehicle (DMSO:NaCl 0.9% 1:1, v:v; 1 ml/kg i.p.). Serum alanine aminotransferase (ALT), plasma and liver triglyceride levels, hepatic malondialdehyde (MDA), reduced glutathione (GSH) concentrations, liver gene expression of Toll-like receptor-4 (TLR-4), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) and intercellular adhesion molecule-1 (ICAM-1) were studied at the end of the study. A histological evaluation of liver damage was also carried out. Raxofelast, an analog of vitamin E, blunted the increased hepatic nuclear factor-ĸB activity, reduced serum ALT, plasma and liver triglycerides, lowered hepatic MDA levels, prevented liver GSH depletion and decreased TLR-4, TNF-α, IL-6 and ICAM-1 hepatic gene expression. Furthermore raxofelast ameliorated liver damage. Our results suggest that raxofelast blunts the inflammatory cascade and organ damage during chronic ethanol exposure.