Vascular permeability factor (VPF) is a 40-kilodalton disulfide-linked dimeric glycoprotein that is active in increasing blood vessel permeability, endothelial cell growth, and angiogenesis. These properties suggest that the expression of VPF by tumor cells could contribute to the increased neovascularization and vessel permeability that are associated with tumor vasculature. The cDNA sequence of VPF from human U937 cells was shown to code for a 189-amino acid polypeptide that is similar in structure to the B chain of platelet-derived growth factor (PDGF-B) and other PDGF-B-related proteins. The overall identity with PDGF-B is 18%. However, all eight of the cysteines in PDGF-B were found to be conserved in human VPF, an indication that the folding of the two proteins is probably similar. Clusters of basic amino acids in the COOH-terminal halves of human VPF and PDGF-B are also prevalent. Thus, VPF appears to be related to the PDGF/v-sis family of proteins.

Nonsteroidal antiinflammatory drugs (NSAIDs) are widely used for the treatment of Inlammator dimseas, but sg t side effects such as g oi l erosion and renal damage lmit their use. NSAIDs Inhibit the enzyme cyclooxygenase (COX), which catalyzes the conversion of arachidonic acid to p (PGs) and thromboxane. Two forms of COX have been identlfled-COX-1, which is constitutively expressed in most tissues and organs, and the Inducible enzyme, COX-2, whih has been localized primarily to ilmmatory cells and tsues. In an animal model of acute inflammation (Injection ofcarrageenan into the footpad), edema was produced that was associated with marked accumulation of COX-2 mRNA and thromboxane. A selective inhibitor of COX-2 (SC-58125) inhibited edema at the inmatory site and was analgesic but had no effect on PG production In the stomach and did not cause gastric toxicity. These data suggest that selective inhibition of COX-2 may produce superior an mmatory drugs with substantial safety advantages over existing NSAIDs. bone density, fluid and electrolyte imbalance, and "Cushinglike" symptoms. These untoward effects limit glucocorticoid use in chronic inflammatory disorders such as rheumatoid arthritis (13).Studies were designed to evaluate the role ofCOX-2 in vivo at the site of inflammation. We report that in a model of inflammation useful in the characterization of NSAIDs, the carrageenan-injected rat paw, COX-2 was expressed locally in response to the proinflammatory stimulus and that the induction of COX-2 mRNA coincided with the synthesis of proinflammatory PGs and the development of edema and hyperalgesia. COX-1 mRNA was detectable in the normal rat paw, but its expression did not change following the onset of the inflammatory reaction. Furthermore, a selective inhibitor of COX-2 {SC-58125: 1-[(4-methylsulfonyl)phenyll-3-trifluoromethyl-5-(4-fluorophenyl)pyrazole} blocked edema and hyperalgesia in vivo following an inflammatory insult, without causing gastric mucosal damage.

We have examined the role of cyclooxygenase 2 (COX-2) in a model of inflammaion in vivo. Carrageenan administration to the subcutaneous rat air pouch induces a rapid inflammatory response characterized by hih levels of prostaglandins (PGs) and leukotrienes in the fluid exudate. The time course of the induction of COX-2 mRNA and protein coincided with the production of PGs in the pouch tissue and cellular infiltrate. Carrageenan-induced COX-2 immunoreactivity was localized to macrophages obtained from the fluid exudate as well as to the inner surface layer of cells within the pouch lining. Dexamethasone inhibited both COX-2 expression and PG synthesis in the fluid exudate but failed to inhibit PG synthesis in the stomach. Furthermore, NS-398, a selective COX-2 inhibitor, and indomethacin, a nonselective COX-1/COX-2 inhibitor, blocked proinflammatory PG synthesis in the air pouch. In contrast, only indomethacin blocked gastric PG and, additionally, produced gastric lesions. These results suggest that inhibitors of COX-2 are potent antiinflammatory agents which do not produce the typical side effects (e.g., gastric ulcers) associated with the nonselective, COX-1-direc antiinfla tory drugs.Nonsteroidal antiinflammatory drugs (NSAIDs) are used to treat acute and chronic inflammatory disorders such as rheumatoid arthritis. The antiinflammatory mechanism of NSAIDs is due to a reduction ofprostaglandin (PG) synthesis by the direct inhibition of cyclooxygenase (COX; prostaglandin-endoperoxide synthase, EC 1.14.99.1) (1). Unfortunately, inhibition of PG production in organs such as stomach and kidney can result in gastric lesions, nephrotoxicity, and increased bleeding.COX exists in two forms. COX-1 is in most tissues and is involved in the physiological production of PGs. COX-2 is cytokine-inducible and is expressed in inflammatory cells (2-9). The identification of constitutive and inducible COX enzymes led to the hypothesis that COX-2 is primarily responsible for PGs produced in inflammation and COX-1 for PGs involved in normal homeostasis (4-6, 10, 11).The rat air pouch is a convenient model to study acute inflammation (12). It is formed by the subcutaneous injection of air over several days and is composed of a lining of cells that consists primarily of macrophages and fibroblasts. Injection of carrageenan into the fully formed air pouch produces an inflammatory granulomatous reaction characterized by a marked production of biochemical mediators in the fluid exudate, including PGs and leukotrienes, as well as a significant influx of polymorphonuclear leukocytes (PMNs) and macrophages (13). Using molecular and pharmacological reagents, we studied the role of COX-2 in this model of inflammation by specific examination of the induction of COX-2 mRNA and protein as well as the production of PGs in the pouch exudate. The results indicate that induction of COX-2 is responsible for the production of PGs at the site of inflammation, whereas the normal synthesis of PGs in the stomach appears to depend on constitutive...

Prostaglandins formed by the cyclooxygenase (COX) enzymes are important mediators of inflammation in arthritis. The contribution of the inducible COX-2 enzyme to inflammation in rat adjuvant arthritis was evaluated by characterization of COX-2 expression in normal and arthritic paws and by pharmacological inhibition of COX-2 activity. The injection of adjuvant induced a marked edema of the hind footpads with coincident local production of PGE 2 . PG production was associated with upregulation of COX-2 mRNA and protein in the affected paws. In contrast, the level of COX-1 mRNA was unaffected by adjuvant injection. TNF-␣ and IL-6 mRNAs were also increased in the inflamed paws as was IL-6 protein in the serum. Therapeutic administration of a selective COX-2 inhibitor, SC-58125, rapidly reversed paw edema and reduced the level of PGE 2 in paw tissue to baseline. Interestingly, treatment with the COX-2 inhibitor also reduced the expression of COX-2 mRNA and protein in the paw. Serum IL-6 and paw IL-6 mRNA levels were also reduced to near normal levels by SC-58125. Furthermore, inhibition of COX-2 resulted in a reduction of the inflammatory cell infiltrate and decreased inflammation of the synovium. Notably, the antiinflammatory effects of SC-58125 were indistinguishable from the effects observed for indomethacin. These results suggest that COX-2 plays a prominent role in the inflammation associated with adjuvant arthritis and that COX-2 derived PGs upregulate COX-2 and IL-6 expression at inflammatory sites. ( J. Clin. Invest. 1996. 97:2672-2679.)

NF-B-induced gene expression contributes significantly to the pathogenesis of inflammatory diseases such as arthritis. I B kinase (IKK) is the converging point for the activation of NF-B by a broad spectrum of inflammatory agonists and is thus a novel target for therapeutic intervention. We describe a small molecule, selective inhibitor of IKK-2, SC-514, which does not inhibit other IKK isoforms or other serine-threonine and tyrosine kinases. SC-514 inhibits the native IKK complex or recombinant human IKK-1/IKK-2 heterodimer and IKK-2 homodimer similarly. IKK-2 inhibition by SC-514 is selective, reversible, and competitive with ATP. SC-514 inhibits transcription of NF-B-dependent genes in IL-1␤-induced rheumatoid arthritis-derived synovial fibroblasts in a dosedependent manner. When the mechanism of NF-B activation was evaluated in the presence of this inhibitor, several interesting observations were found. First, SC-514 did not inhibit the phosphorylation and activation of the IKK complex. Second, there was a delay but not a complete blockade in I B␣ phosphorylation and degradation; likewise there was a slightly slowed, decreased import of p65 into the nucleus and a faster export of p65 from the nucleus. Finally, both I B␣ and p65 were comparable substrates for IKK-2, with similar K m and K cat values, and SC-514 inhibited the phosphorylation of either substrate similarly. Thus, the effect of SC-514 on cytokine gene expression may be a combination of inhibiting I B␣ phosphorylation/degradation, affecting NF-B nuclear import/ export as well as the phosphorylation and transactivation of p65.