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P286 Effects of hypoxia on interleukin-1α-induced peroxynitrite production and articular cartilage matrix metabolism
O s t e o a r t h r i t i s a n d C a r t i l a g e Vol. 13, S u p p l e m e n t A
A)
I
12 I
10 > 4~
1062 Cd-rap 1062 MT Cd-rap
l
ill
II
=1
8
6
• 2 e~
i
B)
1.
Sex9 2.
3.
I
zo% o :
I
I •
,
I
!1.-10~ SO]) +SOD Fig. 1. Effect of hypoxia and SOD on IL-10~-inducedperoxynitrite production as measured by 3-nitrotyrosine immunoblot. Band shown is at 80 kDa.
4
ve~or
1%o,
C o r m ' e l IL-le~
.J
•> 4~
$143
p300 4. ---189 bp
site 1
---DNA control
assay. Statistical significance was determined by ANOVA with Duncan's post-hoc comparison. Results: Hypoxia decreased IL-1o~-induced peroxynitrite formation (Fig. 1). SOD inhibited I L-1o~-induced peroxynitrite formation in both 1% 02 and 20% 02 (Fig, 1). In 1% 02, but not in 20% 02, SOD caused significant inhibition of I L-1o~-induced proteoglycan degradation (Fig. 2). IL-1o~decreased both proteoglycan and collagen II synthesis in cartilage explants in 1% 02 and 20% 02, which was not inhibited by SOD. 40
3S
---169 bp site 2
---DNA control lane ip IL-1I~ TNF-Ix
1 IqG
2 3 4 C/EBP~ C/EBPI~ C/EBP# + +
30 = 25 E lg IO 5 0
C/EBP -2.2
C/EnBP
r,,,,,,,~ Cd_r ap
I
I
Conb'ol
SOD
IL-I~
-2.0
(site 1)
169 bp (site 2)
40 b
i'
3O
may provide clues to treatment of articular joint disease such as osteoarthritis in adult humans.
= 2.!. E
10
P286
5
EFFECTS OF HYPOXIA ON I N T E R L E U K I N - I . - I N D U C E D PEROXYNITRITE P R O D U C T I O N A N D A R T I C U L A R C A R T I L A G E MATRIX METABOLISM
0
J Cernanec 1, F Guilak 1, JB Weinberg 2, I Batinic-Haberle 3, B Fermor 1
1Division of Orthopaedic Surgery, Duke University Medical Center, Durham, NC; 2Department of Medicine, VA Medical Center, Durham, NC; 3Department of Radiation Oncology, Duke University Medical Center, Durham, NC Aim of Study: Elevated levels of nitric oxide (NO) are produced in osteoarthritic cartilage and are associated with cartilage degradation. NO reacts with superoxide anion (02-) to form peroxynitrite, which may modulate cartilage degradation. Oxygen tension in arthritic joints may reach below 1% 02, and low oxygen could influence the formation of superoxide anion or peroxynitrite. The aim of this study was to determine whether hypoxia affects interleukin-1-o~-induced peroxynitrite production and articular cartilage matrix metabolism. Methods: Porcine cartilage explants and chondrocytes seeded in alginate were incubated for 72 hours in either 1% 02 or 20% 02 in media supplemented with either 1 ng/mL interleukin-1o~ (IL100, 25 I~M superoxide dismutase (SOD) mimetic, an antioxidant to scavenge superoxide anion and inhibit peroxynitrite formation, or 1 ng/mL IL-1o~ + 25 I~M SOD mimetic. Peroxynitrite formation was measured by immunoblot with anti-3-nitrotyrosine antibody. Proteoglycan and collagen II synthesis rates were determined by 35S-sulfate and 3H-proline incorporation respectively, and proteoglycan release into the culture media by dimethylmethylene blue
I
lL-lu +GOD
n
mm SOD
! I m
IL-I~
m
IL-Iu +SO0 Fig. 2. Effect of peroxynitrite on IL-10~-inducedproteoglycan degradation at 1% 02 (a) and 20% 02 (b). Mean ± SEM. n > 7 explants from 3 pigs. *p < 0.001, **p < 0.05. Control
Conclusion: Our findings show that hypoxia modulates IL-1o~ induced peroxynitrite production, which in turn influences I L-1o~induced proteoglycan degradation in the presence of the antioxidant SOD. Oxygen tension may have important implications in the biological actions of superoxide anion, NO, and its derivatives, making these inflammatory mediators potential targets in the pharmacologic treatment of osteoarthritis.
P287 INTERLEUKIN-113 IMPAIRS TGFI~ S I G N A L I N G IN A R T I C U L A R C H O N D R O C Y T E S BY D O W N - R E G U L A T I N G TI~RII R E C E P T O R A N D U P R E G U L A T I N G INHIBITORY SMAD7
C Bauge, JM Elissalde, S Leclercq, SJ Kim, JP Puiol, K Boumediene
Laboratory of Connective Tissue, Faculty of Medicine, Caen, France; Department of Anatomy, Faculty of Medicine, Caen, France; Department of Orthopedic Surgery, Clinique St Martin, Caen, France; Laboratory of Chemoprevention, National Cancer Institute, Bethesda, MD Aim: To determine the effect of Interleukin 11~, a key cytokine in
A)
I
12 I
10 > 4~
1062 Cd-rap 1062 MT Cd-rap
l
ill
II
=1
8
6
• 2 e~
i
B)
1.
Sex9 2.
3.
I
zo% o :
I
I •
,
I
!1.-10~ SO]) +SOD Fig. 1. Effect of hypoxia and SOD on IL-10~-inducedperoxynitrite production as measured by 3-nitrotyrosine immunoblot. Band shown is at 80 kDa.
4
ve~or
1%o,
C o r m ' e l IL-le~
.J
•> 4~
$143
p300 4. ---189 bp
site 1
---DNA control
assay. Statistical significance was determined by ANOVA with Duncan's post-hoc comparison. Results: Hypoxia decreased IL-1o~-induced peroxynitrite formation (Fig. 1). SOD inhibited I L-1o~-induced peroxynitrite formation in both 1% 02 and 20% 02 (Fig, 1). In 1% 02, but not in 20% 02, SOD caused significant inhibition of I L-1o~-induced proteoglycan degradation (Fig. 2). IL-1o~decreased both proteoglycan and collagen II synthesis in cartilage explants in 1% 02 and 20% 02, which was not inhibited by SOD. 40
3S
---169 bp site 2
---DNA control lane ip IL-1I~ TNF-Ix
1 IqG
2 3 4 C/EBP~ C/EBPI~ C/EBP# + +
30 = 25 E lg IO 5 0
C/EBP -2.2
C/EnBP
r,,,,,,,~ Cd_r ap
I
I
Conb'ol
SOD
IL-I~
-2.0
(site 1)
169 bp (site 2)
40 b
i'
3O
may provide clues to treatment of articular joint disease such as osteoarthritis in adult humans.
= 2.!. E
10
P286
5
EFFECTS OF HYPOXIA ON I N T E R L E U K I N - I . - I N D U C E D PEROXYNITRITE P R O D U C T I O N A N D A R T I C U L A R C A R T I L A G E MATRIX METABOLISM
0
J Cernanec 1, F Guilak 1, JB Weinberg 2, I Batinic-Haberle 3, B Fermor 1
1Division of Orthopaedic Surgery, Duke University Medical Center, Durham, NC; 2Department of Medicine, VA Medical Center, Durham, NC; 3Department of Radiation Oncology, Duke University Medical Center, Durham, NC Aim of Study: Elevated levels of nitric oxide (NO) are produced in osteoarthritic cartilage and are associated with cartilage degradation. NO reacts with superoxide anion (02-) to form peroxynitrite, which may modulate cartilage degradation. Oxygen tension in arthritic joints may reach below 1% 02, and low oxygen could influence the formation of superoxide anion or peroxynitrite. The aim of this study was to determine whether hypoxia affects interleukin-1-o~-induced peroxynitrite production and articular cartilage matrix metabolism. Methods: Porcine cartilage explants and chondrocytes seeded in alginate were incubated for 72 hours in either 1% 02 or 20% 02 in media supplemented with either 1 ng/mL interleukin-1o~ (IL100, 25 I~M superoxide dismutase (SOD) mimetic, an antioxidant to scavenge superoxide anion and inhibit peroxynitrite formation, or 1 ng/mL IL-1o~ + 25 I~M SOD mimetic. Peroxynitrite formation was measured by immunoblot with anti-3-nitrotyrosine antibody. Proteoglycan and collagen II synthesis rates were determined by 35S-sulfate and 3H-proline incorporation respectively, and proteoglycan release into the culture media by dimethylmethylene blue
I
lL-lu +GOD
n
mm SOD
! I m
IL-I~
m
IL-Iu +SO0 Fig. 2. Effect of peroxynitrite on IL-10~-inducedproteoglycan degradation at 1% 02 (a) and 20% 02 (b). Mean ± SEM. n > 7 explants from 3 pigs. *p < 0.001, **p < 0.05. Control
Conclusion: Our findings show that hypoxia modulates IL-1o~ induced peroxynitrite production, which in turn influences I L-1o~induced proteoglycan degradation in the presence of the antioxidant SOD. Oxygen tension may have important implications in the biological actions of superoxide anion, NO, and its derivatives, making these inflammatory mediators potential targets in the pharmacologic treatment of osteoarthritis.
P287 INTERLEUKIN-113 IMPAIRS TGFI~ S I G N A L I N G IN A R T I C U L A R C H O N D R O C Y T E S BY D O W N - R E G U L A T I N G TI~RII R E C E P T O R A N D U P R E G U L A T I N G INHIBITORY SMAD7
C Bauge, JM Elissalde, S Leclercq, SJ Kim, JP Puiol, K Boumediene
Laboratory of Connective Tissue, Faculty of Medicine, Caen, France; Department of Anatomy, Faculty of Medicine, Caen, France; Department of Orthopedic Surgery, Clinique St Martin, Caen, France; Laboratory of Chemoprevention, National Cancer Institute, Bethesda, MD Aim: To determine the effect of Interleukin 11~, a key cytokine in