Objective. Stromal cell-derived factor 1 (SDF-1; or, CXCL12) is a potent chemotactic and angiogenic factor that has been proposed to play a role in the recruitment of lymphocytes into rheumatoid arthritis (RA) synovium. We tested the hypothesis that synovial SDF-1 expression is regulated by cytokine and hypoxic stimulation, the latter being mediated by hypoxiainducible factor 1␣ (HIF-1␣). These factors regulate the expression of vascular endothelial growth factor (VEGF), itself an important angiogenic mediator.Methods. RA and osteoarthritic synovial fibroblasts and whole tissue explants were cultured under normoxic or hypoxic (1% O 2 ) conditions for up to 72 hours in the presence or absence of interleukin-1␤ (IL-1␤), tumor necrosis factor (TNF), or transforming growth factor ␤ (TGF␤). Expression of HIF-1␣, VEGF, and SDF-1 was detected in synovial tissue and cells by immunohistochemistry and Western blotting. VEGF and SDF-1 expression by cultured synovial fibroblasts was evaluated by reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay.Results. Immunohistochemistry revealed the presence of HIF-1␣, VEGF, and SDF-1 in RA synovium. Patchy expression of HIF-1␣ was detected primarily in the synovial lining and sublining areas; expression in synovial fibroblasts and in the lining cells of whole synovial tissue explants was markedly augmented by hypoxic culture conditions. Hypoxia enhanced the expression of VEGF and SDF-1 messenger RNA in synovial fibroblasts. The production of VEGF and SDF-1 protein by synovial fibroblasts was augmented by 50% and 132%, respectively, after 24 hours of hypoxia. VEGF production was potently induced by TGF␤, and to a lesser extent by IL-1␤ and TNF, and was further augmented by hypoxia. In contrast, none of the tested cytokines induced SDF-1 production.Conclusion. As with VEGF, SDF-1 expression is induced by hypoxia; however, cytokines induce VEGF but not SDF-1. Hypoxic conditions in RA synovium, which are likely to be transient and episodic, may contribute to the persistence of synovitis by inducing VEGF and SDF-1.

Modification of low density lipoprotein (LDL) by free radical oidat renders this molecular complex cytotoxic. Oxidized lipoproteins exist in vivo In atherosclerotic lesions and in the plasma of diabetic i , suggesting that Upoprotein-induced tissue damage may occur in cen diseases. We undertook purifiation and Identicati of the major cytotoxin In oxidized LDL. The lipid extract from oxidized LDL was subjected to multiple HPLC separations, and the fractions were assayed for cytotoxicity. Mass spectrometry and nuclear magnetic resonance Identified the purified toxin as 7p-hydroperoxycholest-5-en-3I3-ol Vascular cells are susceptible to the toxic effects of low density lipoprotein (LDL) or very low density lipoprotein (VLDL) after modification of the lipoproteins by free radical oxidation (1-4). Oxidation of LDL leads to cytotoxin formation whether oxidation is mediated by lipoxygenases (5), metal ions (6), or ultraviolet irradiation (7) in cell-free systems or by the action of free radicals from cultured endothelial cells (8), vascular smooth muscle cells (8), neutrophils (9), or stimulated human monocytes (10). The cytotoxic moiety of oxidized LDL (oxLDL) is extractable with organic solvents (4), and numerous candidate substances could be proposed to explain its toxic action (11).Oxidized lipoproteins occur in vivo in vascular lesions (12)(13)(14) and in plasma of certain diabetic subjects (15). OxLDL has been proposed to play a causal role in atherosclerosis (11,(16)(17)(18) is an important step toward testing evolving theories of atherogenesis that include lipoprotein oxidation (11,(16)(17)(18) and vascular injury (20,21) as putative early events.by dialysis against 2-6 /uM cupric sulfate for various times (23). OxLDL preparations were dialyzed against 0.15 M NaCl/0.5 mM EDTA, pH 8.5, to remove cupric ions. Relative oxidation, measured as thiobarbituric acid reactivity (6,24), was equivalent to 4-6 nmol of malondialdehyde (MDA) per mg of LDL cholesterol.Human foreskin fibroblasts were plated in a 1:1 (vol/vol) mixture of Dulbecco's minimal essential medium and Ham's F-12 medium (DME/F-12) supplemented with 5% (vol/vol) fetal bovine serum (6) Aliquots of5 mg ofnative LDL or oxLDL were lyophilized overnight, and the lipid was extracted with 5 ml of acetone. The mixture was sonicated for 10-20 sec, mixed for 1 min, and allowed to stand for 20 min. After centrifugation at 1000x g for 10-20 min, the residue was reextracted twice, as above. Preliminary experiments revealed that the recovery of the cytotoxic activity by this extraction procedure was equivalent or superior to other means, including chloroform/ methanol extraction ofaqueous lipoprotein solutions. Pooled extracts were dried under nitrogen and redissolved in isopropanol/acetonitrile, 1:1 (vol/vol). After centrifugation for 5 min at 1000 x g, the supernatant was analyzed by reversephase HPLC (Waters 1LBondapak C1s preparative column).The solvent gradient for elution consisted of water/ acetonitrile, 1:1 (vol/vol), which was increased over 5 ...

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