- Email: [email protected]
Time course of oxidative stress after vascular injury in rabbits
I 469 I WMOR
NECROSIS
I 470 RECEPTOR
DEFICIENCY
EXACERBATES
CARDIAC
A&i&& (ADR)’ is a widely used chemotherapeutic agent, but elicits lethal cardiotoxic side effects limiting its cliical application. Recent clinical observations indicate that tumor necrosis factor-alpha (TNPa) may be a major mediator in dierent forms of cardiac injury induced by various agents end pethologicalconditions.Studieshave shownthat MasOD is one of the proteins responsiblefor protectionof tissuesagainstdamagemediatedby TNFa, sad mitochonddamsy be the cellular locuswhere TNFa cytototicity is triggered. Accordingly, we hypothesizedthat TNFa is a mediator in ADR-induced cardiac injury. The purpose of the present study is to determine the roles of TNFaand MaSOD in ADR-induced cardiotoxicity, using transgenic and knockout mice. Six different lines of mice were utilized for experiments including: (1) TNF receptor knockout (DKO) homozygoos, (2) DKO heterozygous, (3) MnSOD overexpression (TgM) homozygoos, (4) Tgh4 heterozygoos, (5) DKO and Tgh4 cross bred (DKOffgM), and (6) wild-type mice. Animals were irjectcd with a single dose of ADR (20&g, ip.) and killed 5 days after the injection Tissue injury was examined by electron ndcro%opy. Oxidative products, 4-HNE and carbonyl proteins, wcrc detected by immunohistochemistty and immtmobtot,respectively.The resultsshowedthat ADR caused morphologicat changes end ultrastructurallesions including cytoplasmic swelling, myelin figares and focal injury within mitochondria of cardiac tissue. The injnry was found to be more severe in the DKO mice, while there were no significant alterations observed in TgM and DKO/TgM mice. The basal levels of antioxidant enzyme activities were similar in all animals tested except for the higher level of MnSOD activity in Tgh4 and DKOmgM mice. However, oxidative products were increased tier ADR treatment. DKO mice exhibited the highest levels of 4-HNE and total carbonyl proteins compared with other groups. Overexpression of MnSOD in DKO mice prevented the ox&live damage caused by ADR Overall, the results indicate that complete blockage of TNFa action may be clinically detrimental. But, a combination of TNFa blockage and MnSOD overexpression may be beneticial.
(1
TIME COURSE OF OXIDATIVE STRESS AFTER VASCULAR INJURY IN RABBITS W CP Azev&Liliete C Souza,Hernldo P Soura,Mariano
]aniszewski,Protnsio L Luz,Froncisco RM LaurindaEmergency and Heart htitute,U.of Sao Paulo,Brazil
Medicine
Dept.
Vascular response to angioplasty underlies a high incidence of restenosis. Our previous work showed massive oxidant stress immediately after vascular balloon injury, in a way proportional to the degree of injury, and accompanied by redox-dependent NF-kappaB activation. However, the time course of vascular redox state during the repair reaction is unknown. Rabbits were submitted to iliac artery overdistension injury and killed immediately or after 7,14 or 28 days. In several groups of experiments (n=14 to 34), we studied the following variables: 1)Neointimal size (intima/media [I/M] area ratio); 2)Cell proliferation index (cells immunostained for PCNA/mm’); 3)Vascular caliber at arteriography (delta%, injured artery vs contralateral control); 4)Lucigenin reductase activity of vascular rings ex vivo (Luc.R., an indirect index of superoxide production, in cpmxlOO/min/mg); 5)Vascular levels of reduced and oxidized glutathione (GSH/GSSG ratio). Results are mea&EM; * p< 0.05 vs. control: I/M ratio PCNA(+)cel. Caliber Luc.R. GSH/GSSG Time 0.5ti.o 19M 2.2*0.2 Control -0M -01to +30+5* 2070f180’ O.S~t0.2’ Immediate -0iO 7 days 0.1M.l 5.5*0.5* +16*4* 101?29* 1.7fl.3 14 days 0.6+0.1* 2.2*0.4* -15s 27f6 28 days 1.5&0.2* 0.7~tO.O -33+3* 9+4 2.3fl.l Thus, oxidative stress after arterial injury is a temporary event, which peaks immediately after injury and is kept increased in apparent correlation with the early proliferative phase of vascular repair. These data allow the hypothesis that oxidative stress acts as an early dose-dependent modulator of signaling processes/gene program that affect vascular remodeling after injury.
I472
ROLE OF GLUTATHIONE IN LPS-INDUCED PRODUCTION IN A HUMAN MONOCYTIC W. Procter and
I
Susan Ward, Steve Hokell, Tim amble Co Mnson, OH 45040.
CYTOKINE CELL LINE
Engel, and Robert E. Singer.
Since glutathione precursors (e.g. N-acetyl cysteine, NAC) can modify expression of certain cytokines in LPS-stimulated macrophages, it can be hypothesized that changes in cell glutathione levels may mediate a redox signal regulating cytokine expression. The goal of this investigation was to understand the role of glutathione in the LPS-mediated induction of cytokine responses by assessing correlation between cytokine responses (TNF and GM-CSF) and cellular levels of the oxidized and reduced forms of glutathione following LPS activation of THP-1 cells. Initial experiments demonstrated treatment of THP-1 cells with NAC decreased levels of LPS-induced TNF- alpha. In turn, differentiated THP-1 cells were either exposed to BSO (500mM) for 24 hris or incubated in parallel in control culture media. Both cell preparations were then challenged with varying concentrations of LPS, and culture supernatants were collected (6hris) and analyzed (ELISA) for TNF-alpha and GM-CSF. At 30 minutes and 6 hours following LPS challenge the cells were quick frozen for storage. Subsequently, they were washed, lysed, extracted, and analyzed (LC/MS/MS) for levels of GSH and GSSG. The THP-1 cells secreted large quantities of TNF-alpha following LI’S challenge, while GM-CSF production was lower but detectable at 6 hours. BSO treatment markedly decreased (-70%) total cell levels of glutathione and increased the ratio of GSH to GSSG. However, BSO treatment did not substantially change the respective levels of TNF-alpha or GM-CSF production in THP-1 cells for any LI’S concentration tested or at any time point evaluated. In conclusion, the data suggest that changes in cell glutathione levels do not mediate induction of LPS-induced cytokine expression.
OXYGEN
INTESTINAL PERMEABILITY AND WEIGHT (BRIEF NOTE) Helion Povoa Jr., Claudia C. Andrade. Cirley Santos. Denise Pifano and Andrea Mahos. CLINICE - Rua Martins Ferreira. 75. Botafogo. Rio de Janeiro. RJ. Brasil. Zip: 22271 010. Fax: 55215390906. E-mail : [email protected] Recent papers have demonstrated that excessive absorption of intestine is somewhat harmful. Intestinal Permeability (IP) is now considered an important factor in genesis and worsening of several diseases. IP is increased afler use of contraceptives, antiinflammatory agents, cortiwsteroids and antibiotics, as well as in patients with inflammatory bowel disease, spondilttis, extensive burning and food allergy. 31 women were studied thoughout this study. They were divided into 2 (two) groups: one with a Body Mass Index (BMI) greater than 25 (29.7 + 57) and the other with BMI less than 25 (215 i 2.1). Intestinal Permeability was assessed by using the La&nose test. Normal values are less than 1.6 % of absorption. The range of both groups age was between 20-60 years. Data are presented In Table I. Table I - Intestinal Permeability BMI’ Laotulose absorption’ Group I (16 cases) Group II (15 oases)
2937 k 3.7 (’ 25) 21,5 + 2,l (< 25)
2,45 zt I,67 1.34 i 0,475
‘Mean + Standard Deviation The increase of IP in gmup I is statistically significant 0,02.
’ 9
9
t = 2,64;
p<
5155
NECROSIS
I 470 RECEPTOR
DEFICIENCY
EXACERBATES
CARDIAC
A&i&& (ADR)’ is a widely used chemotherapeutic agent, but elicits lethal cardiotoxic side effects limiting its cliical application. Recent clinical observations indicate that tumor necrosis factor-alpha (TNPa) may be a major mediator in dierent forms of cardiac injury induced by various agents end pethologicalconditions.Studieshave shownthat MasOD is one of the proteins responsiblefor protectionof tissuesagainstdamagemediatedby TNFa, sad mitochonddamsy be the cellular locuswhere TNFa cytototicity is triggered. Accordingly, we hypothesizedthat TNFa is a mediator in ADR-induced cardiac injury. The purpose of the present study is to determine the roles of TNFaand MaSOD in ADR-induced cardiotoxicity, using transgenic and knockout mice. Six different lines of mice were utilized for experiments including: (1) TNF receptor knockout (DKO) homozygoos, (2) DKO heterozygous, (3) MnSOD overexpression (TgM) homozygoos, (4) Tgh4 heterozygoos, (5) DKO and Tgh4 cross bred (DKOffgM), and (6) wild-type mice. Animals were irjectcd with a single dose of ADR (20&g, ip.) and killed 5 days after the injection Tissue injury was examined by electron ndcro%opy. Oxidative products, 4-HNE and carbonyl proteins, wcrc detected by immunohistochemistty and immtmobtot,respectively.The resultsshowedthat ADR caused morphologicat changes end ultrastructurallesions including cytoplasmic swelling, myelin figares and focal injury within mitochondria of cardiac tissue. The injnry was found to be more severe in the DKO mice, while there were no significant alterations observed in TgM and DKO/TgM mice. The basal levels of antioxidant enzyme activities were similar in all animals tested except for the higher level of MnSOD activity in Tgh4 and DKOmgM mice. However, oxidative products were increased tier ADR treatment. DKO mice exhibited the highest levels of 4-HNE and total carbonyl proteins compared with other groups. Overexpression of MnSOD in DKO mice prevented the ox&live damage caused by ADR Overall, the results indicate that complete blockage of TNFa action may be clinically detrimental. But, a combination of TNFa blockage and MnSOD overexpression may be beneticial.
(1
TIME COURSE OF OXIDATIVE STRESS AFTER VASCULAR INJURY IN RABBITS W CP Azev&Liliete C Souza,Hernldo P Soura,Mariano
]aniszewski,Protnsio L Luz,Froncisco RM LaurindaEmergency and Heart htitute,U.of Sao Paulo,Brazil
Medicine
Dept.
Vascular response to angioplasty underlies a high incidence of restenosis. Our previous work showed massive oxidant stress immediately after vascular balloon injury, in a way proportional to the degree of injury, and accompanied by redox-dependent NF-kappaB activation. However, the time course of vascular redox state during the repair reaction is unknown. Rabbits were submitted to iliac artery overdistension injury and killed immediately or after 7,14 or 28 days. In several groups of experiments (n=14 to 34), we studied the following variables: 1)Neointimal size (intima/media [I/M] area ratio); 2)Cell proliferation index (cells immunostained for PCNA/mm’); 3)Vascular caliber at arteriography (delta%, injured artery vs contralateral control); 4)Lucigenin reductase activity of vascular rings ex vivo (Luc.R., an indirect index of superoxide production, in cpmxlOO/min/mg); 5)Vascular levels of reduced and oxidized glutathione (GSH/GSSG ratio). Results are mea&EM; * p< 0.05 vs. control: I/M ratio PCNA(+)cel. Caliber Luc.R. GSH/GSSG Time 0.5ti.o 19M 2.2*0.2 Control -0M -01to +30+5* 2070f180’ O.S~t0.2’ Immediate -0iO 7 days 0.1M.l 5.5*0.5* +16*4* 101?29* 1.7fl.3 14 days 0.6+0.1* 2.2*0.4* -15s 27f6 28 days 1.5&0.2* 0.7~tO.O -33+3* 9+4 2.3fl.l Thus, oxidative stress after arterial injury is a temporary event, which peaks immediately after injury and is kept increased in apparent correlation with the early proliferative phase of vascular repair. These data allow the hypothesis that oxidative stress acts as an early dose-dependent modulator of signaling processes/gene program that affect vascular remodeling after injury.
I472
ROLE OF GLUTATHIONE IN LPS-INDUCED PRODUCTION IN A HUMAN MONOCYTIC W. Procter and
I
Susan Ward, Steve Hokell, Tim amble Co Mnson, OH 45040.
CYTOKINE CELL LINE
Engel, and Robert E. Singer.
Since glutathione precursors (e.g. N-acetyl cysteine, NAC) can modify expression of certain cytokines in LPS-stimulated macrophages, it can be hypothesized that changes in cell glutathione levels may mediate a redox signal regulating cytokine expression. The goal of this investigation was to understand the role of glutathione in the LPS-mediated induction of cytokine responses by assessing correlation between cytokine responses (TNF and GM-CSF) and cellular levels of the oxidized and reduced forms of glutathione following LPS activation of THP-1 cells. Initial experiments demonstrated treatment of THP-1 cells with NAC decreased levels of LPS-induced TNF- alpha. In turn, differentiated THP-1 cells were either exposed to BSO (500mM) for 24 hris or incubated in parallel in control culture media. Both cell preparations were then challenged with varying concentrations of LPS, and culture supernatants were collected (6hris) and analyzed (ELISA) for TNF-alpha and GM-CSF. At 30 minutes and 6 hours following LPS challenge the cells were quick frozen for storage. Subsequently, they were washed, lysed, extracted, and analyzed (LC/MS/MS) for levels of GSH and GSSG. The THP-1 cells secreted large quantities of TNF-alpha following LI’S challenge, while GM-CSF production was lower but detectable at 6 hours. BSO treatment markedly decreased (-70%) total cell levels of glutathione and increased the ratio of GSH to GSSG. However, BSO treatment did not substantially change the respective levels of TNF-alpha or GM-CSF production in THP-1 cells for any LI’S concentration tested or at any time point evaluated. In conclusion, the data suggest that changes in cell glutathione levels do not mediate induction of LPS-induced cytokine expression.
OXYGEN
INTESTINAL PERMEABILITY AND WEIGHT (BRIEF NOTE) Helion Povoa Jr., Claudia C. Andrade. Cirley Santos. Denise Pifano and Andrea Mahos. CLINICE - Rua Martins Ferreira. 75. Botafogo. Rio de Janeiro. RJ. Brasil. Zip: 22271 010. Fax: 55215390906. E-mail : [email protected] Recent papers have demonstrated that excessive absorption of intestine is somewhat harmful. Intestinal Permeability (IP) is now considered an important factor in genesis and worsening of several diseases. IP is increased afler use of contraceptives, antiinflammatory agents, cortiwsteroids and antibiotics, as well as in patients with inflammatory bowel disease, spondilttis, extensive burning and food allergy. 31 women were studied thoughout this study. They were divided into 2 (two) groups: one with a Body Mass Index (BMI) greater than 25 (29.7 + 57) and the other with BMI less than 25 (215 i 2.1). Intestinal Permeability was assessed by using the La&nose test. Normal values are less than 1.6 % of absorption. The range of both groups age was between 20-60 years. Data are presented In Table I. Table I - Intestinal Permeability BMI’ Laotulose absorption’ Group I (16 cases) Group II (15 oases)
2937 k 3.7 (’ 25) 21,5 + 2,l (< 25)
2,45 zt I,67 1.34 i 0,475
‘Mean + Standard Deviation The increase of IP in gmup I is statistically significant 0,02.
’ 9
9
t = 2,64;
p<
5155