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Lipid peroxidation stimulates collagen synthesis in human dermal fibroblasts
4
Oxidative Stress Genes and Proteins
PEROXIDATION STIMULATES COLLAGEN 1.13 LIPID SYNTHESIS IN HUMAN DERMAL FIBROBLASTS Jeffrey^C. Geesin, L2 L a u r ~ H e n d r i c k s , 2 j o e l S. Gordon, 2 and1 Richard A. Berg~ Graduate Program in Biochemistry, Rutgers University and the University of Medicine and Dentistry.of New Jersey-Robert Wood Johnson Medical School, j Piscataway, New Je~ey and Johnson and Johnson Consumer Products, lnc., ~ Skillman, New Jersey. Ascorbate (50-200 ~M) has been shown to stimulate collagen synthesis in cultured human dermal fibroblasts by increasing transcription of the collagen genes. Since ascorbate causes lipid peroxidation in an iron dependent reaction, we tested inhibitors of lipid peroxidation on ascorbate stimulated collagen synthesis. Traditional lipid peroxidation inhibitors, such as propyl gallate, cobalt chloride, and ~-naphthol, inhibit ascorbate-stimulated collagen synthesis and lipid peroxidation at similar c o n c e n t r a t i o n s for both effects. Mannitol, a water soluble anti-oxidant, has no effect on ascorbate-stimulated collagen synthesis and lipid peroxidation, while ~-tocopherol and natural retinoids inhibit both, indicating that lipophilic anti-oxidants can prevent these responses. Superoxide dismutase, Catalase, and their polyethylene glycol conjugates did not inhibit the ascorbate stimulated collagen synthesis. These results suggest a correlation between collagen synthesis and lipid p e r o x i d a t i o n and suggest an explanation for the fibrosis occurring in oxidant induced tissue injury.
1.15 DIFFERENTIAL SENSITIVITY OF
E N D O T H E L I A L CELLS TO THE EFFECTS OF MM-LDL. S.Imes, A. Andalibi, F. Parhami, S. Lavi,S. Lee, A.J. Lusis, J.A. Berliner, M.C.Territo, M.Navab and A.M. Fogelman UCLA School of Medicine, Los Angeles CA.90024 Endothelial cells (EC) from rabbit aorta, human umbilical vein (HUV) and human aorta all can be induced to bind monocytes by minimally oxidized LDL (MM-LDL). However the dose response curve, and the time course of the this effect differ between these cell types and between cells from individual donors. Cells exposed to low levels of MM-LDL become resistant to the effects of higher levels and cycloheximide increases their sensitivity, thus implying that a rapidly turning over protein may be confering resistance to the effects of MM-LDL.An important role for manganese dependent superoxxde dismutase (MnSOD) is suggested by the followin~ observations: Rabbit aortic EC are v e ~ sensitive to high levels of MM-LDL and show little Induction of MnSOD upon acute exposure. However, exposure of rabbit EC to low levels of MMLDL over a period of 2 days leads to an increase in message for MnSOD and a subsequent increase in activity of the enzyme in the cells. This exposure makes the cells resistant to high levels of MM-LDL. In contrast, H U V are relatively resistant to MM-LDL, and major increases of message for MnSOD are induced on acute exposure to high levels MM-LDL. These results suggest that MnSOD acts to remove the oxlgen free radicals formed in response to minimally omdized lipids and thus MnSOD may play an important role in preventing endothehal activation by these radicals.
ARE MANGANESE AND IRON SUPEROXIDE DISMUTASES OF ESCHERICHIA COLI K-12 PHYSIOLOGICALLY EQUIVALENT'? Karen A. Hopkin and Howard M. Steinman Department of Biochemistry, Albert Einstein College of Medicine, Bronx, NY 10461 U.S.A.
1.14
E. coli contains two highly homologous sui~eroxide
dismutases, one containing manganese (MnSuD) and the other iron (FeSOD), that catalyze the dismutation of superoxide with comparable efficiency. Although MnSOD and FeSOD are distinct in their regulation in response to oxidative stress, it is not known if they act in an equivalent fashion in protecting E. coli from oxygen toxicity. To address this issue, we have developed a system for expressing exclusively Mn or FeSOD in otherwise isogenlc E. coil strains, in which SOD activity can be controlled by culture conditions independent of oxidative stress. The endogenous promoter of each SOD gene was removed by digestion with exonuclease III and replaced with a tac promoter by cloning the truncated ~gene into a plasmid that allows tight regulation o t sod expression. Each construction was introduced into an E. coli mutant deleted in both SOD genes. Using selection on plates containing paraquat and isopropyl-~-D-thiogalactoside (IPTG), we isolated strains expressing either Mn or FeSOD. In each strain the SOD activity units per mg of cell protein increases in proportion to [IPTG] in the medium. At equal SOD activity, the MnSOD strain grows more rapidly aerobically and is more tolerant to paraquat stress than the FeSOD strain, in rich medium on plates or in liquid culture. Studies are in progress to evaluate our initial results that suggest Mn and FeSOD may not be equivalent in vivo.
TI~ SlfiNIFIf.q~ OF FREERADICALS ~ LIPID FflZOXIDATIUN IN DAMA~TOE R ~ AFTI~~ INJI.RII~IN RATS Xia~in9 ~en,~ Zongrong Chen Department of Rediatlon Medicine, Third Nilitary Medical College, Chongqing630038, China Thermal burnln,q on the back of rats (20N TI~ futt-thiclmess) was used to study the changes of free radicals, lipid peroxldatlon and antio~idant function of erTthrocytes after burn injuries.After burn injuries, a large ammmtof lipid peroxides (120) was produced in the skin. This might be transfered into bloud. Free radicals might increase and induce the elevation of plasma Ll~, reduce the activity of plasma 9tutathlone perc~idase (G~px) and the content of ptasm vitamin E, The activity of ~ G~llpx increased si~tificantty on day I posthurn. Ken RBC C~ffpxcould not reverse the effect of injury, the function of antioxidation, such as the activities of ~ G,~px and concentration of G~J, NPSH and vitamin E decreasd. RBC120 incresed, which, cross-linked with sutfl~'dzTl proteins, caused elevation of l~-induced hemolysis, and resulted in severe dunage to RBCmembranes. The results were proved by the experimants in vitro. It can be concluded that the zechanism of damage of RBC after burn injuries is due to a chain reaction of free radicals and lipid per~idation of membranes. It also proves that there are chanoes of functional capability of antioxidation in vivo after burn injuries. Further study is needed to locate the site of production of free radicals within or,~md around P,~C.
1.16
Oxidative Stress Genes and Proteins
PEROXIDATION STIMULATES COLLAGEN 1.13 LIPID SYNTHESIS IN HUMAN DERMAL FIBROBLASTS Jeffrey^C. Geesin, L2 L a u r ~ H e n d r i c k s , 2 j o e l S. Gordon, 2 and1 Richard A. Berg~ Graduate Program in Biochemistry, Rutgers University and the University of Medicine and Dentistry.of New Jersey-Robert Wood Johnson Medical School, j Piscataway, New Je~ey and Johnson and Johnson Consumer Products, lnc., ~ Skillman, New Jersey. Ascorbate (50-200 ~M) has been shown to stimulate collagen synthesis in cultured human dermal fibroblasts by increasing transcription of the collagen genes. Since ascorbate causes lipid peroxidation in an iron dependent reaction, we tested inhibitors of lipid peroxidation on ascorbate stimulated collagen synthesis. Traditional lipid peroxidation inhibitors, such as propyl gallate, cobalt chloride, and ~-naphthol, inhibit ascorbate-stimulated collagen synthesis and lipid peroxidation at similar c o n c e n t r a t i o n s for both effects. Mannitol, a water soluble anti-oxidant, has no effect on ascorbate-stimulated collagen synthesis and lipid peroxidation, while ~-tocopherol and natural retinoids inhibit both, indicating that lipophilic anti-oxidants can prevent these responses. Superoxide dismutase, Catalase, and their polyethylene glycol conjugates did not inhibit the ascorbate stimulated collagen synthesis. These results suggest a correlation between collagen synthesis and lipid p e r o x i d a t i o n and suggest an explanation for the fibrosis occurring in oxidant induced tissue injury.
1.15 DIFFERENTIAL SENSITIVITY OF
E N D O T H E L I A L CELLS TO THE EFFECTS OF MM-LDL. S.Imes, A. Andalibi, F. Parhami, S. Lavi,S. Lee, A.J. Lusis, J.A. Berliner, M.C.Territo, M.Navab and A.M. Fogelman UCLA School of Medicine, Los Angeles CA.90024 Endothelial cells (EC) from rabbit aorta, human umbilical vein (HUV) and human aorta all can be induced to bind monocytes by minimally oxidized LDL (MM-LDL). However the dose response curve, and the time course of the this effect differ between these cell types and between cells from individual donors. Cells exposed to low levels of MM-LDL become resistant to the effects of higher levels and cycloheximide increases their sensitivity, thus implying that a rapidly turning over protein may be confering resistance to the effects of MM-LDL.An important role for manganese dependent superoxxde dismutase (MnSOD) is suggested by the followin~ observations: Rabbit aortic EC are v e ~ sensitive to high levels of MM-LDL and show little Induction of MnSOD upon acute exposure. However, exposure of rabbit EC to low levels of MMLDL over a period of 2 days leads to an increase in message for MnSOD and a subsequent increase in activity of the enzyme in the cells. This exposure makes the cells resistant to high levels of MM-LDL. In contrast, H U V are relatively resistant to MM-LDL, and major increases of message for MnSOD are induced on acute exposure to high levels MM-LDL. These results suggest that MnSOD acts to remove the oxlgen free radicals formed in response to minimally omdized lipids and thus MnSOD may play an important role in preventing endothehal activation by these radicals.
ARE MANGANESE AND IRON SUPEROXIDE DISMUTASES OF ESCHERICHIA COLI K-12 PHYSIOLOGICALLY EQUIVALENT'? Karen A. Hopkin and Howard M. Steinman Department of Biochemistry, Albert Einstein College of Medicine, Bronx, NY 10461 U.S.A.
1.14
E. coli contains two highly homologous sui~eroxide
dismutases, one containing manganese (MnSuD) and the other iron (FeSOD), that catalyze the dismutation of superoxide with comparable efficiency. Although MnSOD and FeSOD are distinct in their regulation in response to oxidative stress, it is not known if they act in an equivalent fashion in protecting E. coli from oxygen toxicity. To address this issue, we have developed a system for expressing exclusively Mn or FeSOD in otherwise isogenlc E. coil strains, in which SOD activity can be controlled by culture conditions independent of oxidative stress. The endogenous promoter of each SOD gene was removed by digestion with exonuclease III and replaced with a tac promoter by cloning the truncated ~gene into a plasmid that allows tight regulation o t sod expression. Each construction was introduced into an E. coli mutant deleted in both SOD genes. Using selection on plates containing paraquat and isopropyl-~-D-thiogalactoside (IPTG), we isolated strains expressing either Mn or FeSOD. In each strain the SOD activity units per mg of cell protein increases in proportion to [IPTG] in the medium. At equal SOD activity, the MnSOD strain grows more rapidly aerobically and is more tolerant to paraquat stress than the FeSOD strain, in rich medium on plates or in liquid culture. Studies are in progress to evaluate our initial results that suggest Mn and FeSOD may not be equivalent in vivo.
TI~ SlfiNIFIf.q~ OF FREERADICALS ~ LIPID FflZOXIDATIUN IN DAMA~TOE R ~ AFTI~~ INJI.RII~IN RATS Xia~in9 ~en,~ Zongrong Chen Department of Rediatlon Medicine, Third Nilitary Medical College, Chongqing630038, China Thermal burnln,q on the back of rats (20N TI~ futt-thiclmess) was used to study the changes of free radicals, lipid peroxldatlon and antio~idant function of erTthrocytes after burn injuries.After burn injuries, a large ammmtof lipid peroxides (120) was produced in the skin. This might be transfered into bloud. Free radicals might increase and induce the elevation of plasma Ll~, reduce the activity of plasma 9tutathlone perc~idase (G~px) and the content of ptasm vitamin E, The activity of ~ G~llpx increased si~tificantty on day I posthurn. Ken RBC C~ffpxcould not reverse the effect of injury, the function of antioxidation, such as the activities of ~ G,~px and concentration of G~J, NPSH and vitamin E decreasd. RBC120 incresed, which, cross-linked with sutfl~'dzTl proteins, caused elevation of l~-induced hemolysis, and resulted in severe dunage to RBCmembranes. The results were proved by the experimants in vitro. It can be concluded that the zechanism of damage of RBC after burn injuries is due to a chain reaction of free radicals and lipid per~idation of membranes. It also proves that there are chanoes of functional capability of antioxidation in vivo after burn injuries. Further study is needed to locate the site of production of free radicals within or,~md around P,~C.
1.16