Leslie J. Crofford

Cyclooxygenase in biology and disease

Cyclooxygenase (COX), the key enzyme required for the conversion of arachidonic acid to prostaglandins was first identified over 20 years ago. Drugs, like aspirin, that inhibit cyclooxygenase activity have been available to the public for about 100 years. In the past decade, however, more progress has been made in understanding the role of cyclooxygenase enzymes in biology and disease than at any other time in history. Two cyclooxygenase isoforms have been identified and are referred to as COX‐1 and COX‐2. Under many circumstances the COX‐1 enzyme is produced constitutively (i.e., gastric mucosa) whereas COX‐2 is inducible (i.e., sites of inflammation). Here, we summarize the current understanding of the role of cyclooxygenase‐1 and ‐2 in different physiological situations and disease processes ranging from inflammation to cancer. We have attempted to include all of the most relevant material in the field, but due to the rapid progress in this area of research we apologize that certain recent findings may have been left out.—DuBois, R. N., Abramson, S. B., Crofford, L., Gupta, R. A., Simon, L. S., van de Putte, L. B. A., Lipsky, P. E. Cyclooxygenase in biology and disease. FASEB J. 12, 1063–1073 (1998)

Cyclooxygenase-1 and -2 expression in rheumatoid synovial tissues. Effects of interleukin-1 beta, phorbol ester, and corticosteroids.

High levels of immunoreactive cyclooxygenase (Cox; prostaglandin H synthase) are present in synovia from patients with rheumatoid arthritis (RA). We now show that the recently identified inducible isoform of Cox, Cox-2, is expressed in synovia from patients with RA. To further explore modulation of the Cox isoforms in RA synovial tissues, we examined the expression and modulation of Cox-1 and -2 in rheumatoid synovial explant cultures and cultured rheumatoid synovial fibroblast-like cells (synoviocytes). Immunoprecipitation of in vitro labeled proteins and Western blot analysis demonstrated the presence of both Cox-1 and -2 under basal conditions in freshly explanted rheumatoid synovial tissues. De novo synthesis of Cox-2 polypeptide was enhanced by IL-1 beta or PMA, and dramatically suppressed by dexamethasone (dex). Cox-1 expression, under the same conditions, showed only minor variation. Since mRNA for Cox-2 is highly unstable, we examined the regulation of Cox-2 transcripts in cultured rheumatoid synoviocytes. Under basal conditions both Cox-1 and -2 mRNAs were present at low levels, but Cox-2 mRNA was markedly increased by treatment with IL-1 beta or PMA. dex markedly suppressed the induction of Cox-2 mRNA. In sharp contrast, Cox-1 transcripts were not modulated by IL-1 beta or dex. These data suggest that modulation of Cox-2 expression by IL-1 beta and corticosteroids may be an important component of the inflammatory process in synovial tissues from patients with RA.

Induction of vascular endothelial growth factor expression in synovial fibroblasts by prostaglandin E and interleukin‐1: a potential mechanism for inflammatory angiogenesis

Inflammatory mediators such as prostaglandin E2 (PGE2) and interleukin‐1 (IL‐1) induce angiogenesis by yet undefined mechanisms. We demonstrate that PGE2 and IL‐1 induces the expression of vascular endothelial growth factor (VEGF), a selective angiogenic factor by rheumatoid synovial fibroblast cells. Transcripts for the EP1 and EP2, subtypes of PGE receptors are expressed in synovial fibroblasts. Activators of protein kinase A pathway stimulated the expression of VEGF whereas down‐regulation of protein kinase C did not influence the PGE effect, suggesting that signalling from the EP2 receptor via the protein kinase A pathway is important. The induction of VEGF expression by PGE2 and interleukin‐1α a may be an important mechanism in inflammatory angiogenesis.

Cultured lung fibroblasts isolated from patients with idiopathic pulmonary fibrosis have a diminished capacity to synthesize prostaglandin E2 and to express cyclooxygenase-2.

Prostaglandin E2 (PGE2) inhibits fibroblast proliferation and collagen synthesis. In this study, we compared lung fibroblasts isolated from patients with idiopathic pulmonary fibrosis (F-IPF) and from patients undergoing resectional surgery for lung cancer (F-nl) with respect to their capacity for PGE2 synthesis and their expression and regulation of cyclooxygenase (COX) proteins. Basal COX activity, assessed by quantitating immunoreactive PGE2 synthesized from arachidonic acid, was twofold less (P < 0.05) in F-IPF than F-nl. In F-nl, incubation with the agonists PMA, LPS, or IL-1 increased COX activity and protein expression of the inducible form of COX, COX-2, and these responses were inhibited by coincubation with dexamethasone. By contrast, F-IPF failed to demonstrate increases in COX-2 protein expression or COX activity in response to these agonists. Under conditions of maximal induction, COX activity in F-IPF was sixfold less than that in F-nl (P < 0.05). Our data indicate that F-IPF have a striking defect in their capacity to synthesize the antiinflammatory and antifibrogenic molecule PGE2, apparently because of a diminished induction of COX-2 protein. This reduction in the endogenous capacity of F-IPF to down-regulate their function via PGE2 may contribute to the inflammatory and fibrogenic response in IPF. Moreover, we believe that this represents the first description of a defect in COX-2 expression in association with a human disease.

In vivo cyclooxygenase expression in synovial tissues of patients with rheumatoid arthritis and osteoarthritis and rats with adjuvant and streptococcal cell wall arthritis.

Cyclooxygenase (COX), or prostaglandin (PG) H synthase, plays a role in inflammatory diseases, but very limited data exist on the regulation of COX in vivo. We, therefore, studied the in vivo expression of COX in synovia from patients with rheumatoid arthritis (RA) and osteoarthritis (OA), as well as joints of rats with streptococcal cell wall (SCW) and adjuvant arthritis. Extensive and intense intracellular COX immunostaining, which correlated with the extent and intensity of mononuclear cell infiltration, was observed in cells throughout RA synovia. Significantly less or equivocal staining was noted in OA and normal human synovia. Similarly, COX immunostaining was equivocal in the joints of normal and arthritis-resistant F344/N rats. In contrast, high level expression developed rapidly in euthymic female Lewis (LEW/N) rats throughout the hindlimb joints and overlying tissues including skin, preceding or paralleling clinically apparent experimental arthritis. COX was expressed in the joints of athymic LEW.rnu/rnu rats 2-4 d after injection of SCW or adjuvant but was not sustained. Physiological doses of antiinflammatory glucocorticoids, but not progesterone, suppressed both arthritis and COX expression in LEW/N rats. These observations suggest that, in vivo, (a) COX expression is upregulated in inflammatory joint diseases, (b) the level of expression is genetically controlled and is a biochemical correlate of disease severity, (c) sustained high level up-regulation is T cell dependent, and (d) expression is down-regulated by antiinflammatory glucocorticoids.

Microsomal Prostaglandin E Synthase Is Regulated by Proinflammatory Cytokines and Glucocorticoids in Primary Rheumatoid Synovial Cells

The selective induction of PGE2 synthesis in inflammation suggests that a PGE synthase may be linked to an inducible pathway for PG synthesis. We examined the expression of the recently cloned inducible microsomal PGE synthase (mPGES) in synoviocytes from patients with rheumatoid arthritis, its modulation by cytokines and dexamethasone, and its linkage to the inducible cyclooxygenase-2. Northern blot analysis showed that IL-1β or TNF-α treatment induces mPGES mRNA from very low levels at baseline to maximum levels at 24 h. IL-1β-induced mPGES mRNA was inhibited by dexamethasone in a dose-dependent fashion. Western blot analysis demonstrated that mPGES protein was induced by IL-1β, and maximum expression was sustained for up to 72 h. There was a coordinated up-regulation of cyclooxygenase-2 protein, although peak expression was earlier. Differential Western blot analysis of the microsomal and the cytosolic fractions revealed that the induced expression of mPGES protein was limited to the microsomal fraction. The detected mPGES protein was catalytically functional as indicated by a 3-fold increase of PGES activity in synoviocytes following treatment with IL-1β; this increased synthase activity was limited to the microsomal fraction. In summary, these data demonstrate an induction of mPGES in rheumatoid synoviocytes by proinflammatory cytokines. This novel pathway may be a target for therapeutic intervention for patients with arthritis.

Basic biology and clinical application of specific cyclooxygenase‐2 inhibitors

In summary, COX-2 is a highly regulated gene product that catalyzes the local production of PGs in pathologic and physiologic situations (Figure 1). It is clear that COX-2 is the isoform responsible for production of the PGs that mediate inflammation, pain, and fever. However, the role for COX-2 in normal physiology is still being defined. Specific COX-2 inhibitors represent a significant conceptual advance in therapy for patients with arthritis. Although there is no expectation of superior efficacy, clinical trials suggest that efficacy will be comparable with that of nonselective NSAIDs. Clinical trials demonstrate the potential for clinically meaningful reductions in the incidence of the most serious GI complications found with nonselective NSAIDs, i.e., ulcer, perforation, and GI bleeding. Over the next several years, treatment of large numbers of patients with specific COX-2 inhibitors will help to define the biology of COX-2. The magnitude of this advance in the therapy of rheumatic diseases is yet to be accurately determined, but the development of specific COX-2 inhibitors may afford significant new treatment options for many patients.

Fibromyalgia relapse evaluation and efficacy for durability of meaningful relief (FREEDOM): A 6-month, double-blind, placebo-controlled trial with pregabalin

&NA; This was a multicenter, double‐blind (DB), placebo‐controlled, randomized discontinuation trial to evaluate the efficacy of pregabalin monotherapy for durability of effect on fibromyalgia (FM) pain. The trial included a 6‐week open‐label (OL) pregabalin‐treatment period followed by 26‐week DB treatment with placebo or pregabalin. Adults with FM and ⩾40‐mm score on 100‐mm pain visual analog scale (VAS) were eligible. During OL weeks 1–3, patients received escalating dosages of pregabalin to determine their optimal dosages. During OL weeks 4–6, patients received their optimal fixed dosages (300, 450, 600 mg/d). To be randomized, patients must have had ⩾50% decrease in pain VAS and a self‐rating of “much” or “very much” improved on Patient Global Impression of Change (PGIC) at the end of OL. Double‐blind treatment was with placebo or the patient’s optimal fixed dosage of pregabalin. Primary outcome was time to loss of therapeutic response (LTR), defined as <30% reduction in pain (from OL baseline) or worsening of FM. A total of 1051 patients entered OL; 287 were randomized to placebo, 279 to pregabalin. Time to LTR was longer for pregabalin versus placebo (P < .0001). Kaplan–Meier estimates of time‐to‐event showed half the placebo group had LTR by Day 19; half the pregabalin group still had not lost response by trial end. At the end of DB, 174 (61%) placebo patients met LTR criteria versus 90 (32%) pregabalin patients. Pregabalin was well tolerated, though 178 (17%) discontinued during OL for treatment‐related adverse events (AE), and more pregabalin than placebo patients discontinued for AEs during DB. In those who respond, pregabalin demonstrated durability of effect for relieving FM pain.

Nephrogenic fibrosing dermopathy: a novel cutaneous fibrosing disorder in patients with renal failure

BACKGROUND Nephrogenic fibrosing dermopathy is a newly recognized cutaneous fibrosing disorder marked by the acute onset of induration involving the upper and lower limbs in patients with acute or chronic renal failure. The etiology, pathogenesis, associated clinical conditions (other than renal failure), and ultimate course have not been defined in the few cases studied. Presently, there is no effective treatment, and the condition persists in most patients. METHODS Clinical and histopathologic data on 13 patients from our institution with the diagnosis of nephrogenic fibrosing dermopathy were reviewed. Several clinical and laboratory parameters were examined to see if any were consistently associated with the disease. Biopsy specimens were analyzed to determine if there was a pattern to the evolution of fibrosis in these patients. RESULTS All 13 patients had renal failure before disease onset: 8 were undergoing chronic hemodialysis, 2 were undergoing chronic peritoneal dialysis, and 3 with acute renal failure had never undergone dialysis before the development of dermopathy. Most patients had other serious underlying medical conditions. Many patients were taking erythropoietin, cyclosporine, or both before the onset of disease. In transplant patients, no histocompatibility antigens were found to be associated with the disease. There were various laboratory abnormalities, but none were consistently associated with the condition. In skin biopsy specimens taken 7 to 180 days after disease onset, there were histopathologic changes suggestive of a tissue reaction to injury, as well as the development of smooth muscle actin-positive myofibroblasts. CONCLUSION Nephrogenic fibrosing dermopathy is a novel cutaneous fibrosing disorder that is distinguished from other sclerosing or fibrosing skin disorders by distinctive clinical and histopathologic findings occurring in the setting of renal failure. There were no additional clinical risk factors or laboratory findings common to the 13 patients studied, other than renal failure. The resemblance to a tissue injury reaction and the presence of myofibroblasts in the tissue specimens suggest that fibrogenic cytokines may be involved in the evolution of the disease.

Perception of noxious and innocuous heat stimulation among healthy women and women with fibromyalgia: association with mood, somatic focus, and catastrophizing

&NA; Recent studies have demonstrated that persons with fibromyalgia display abnormal processing of different types of painful stimulation, suggesting the disorder is characterized by a central pain‐processing deficit not limited specifically to muscle pain. In the present study, 20 women with fibromyalgia and 20 normal, healthy women were compared on measures of pressure pain stimulation and response to contact thermal heat at both noxious and innocuous intensities. Women with fibromyalgia displayed significantly lower pressure pain thresholds at 18 tender point locations as defined by the American College of Rheumatology criteria, as well as lower pressure pain thresholds at five control sites. Women with fibromyalgia had significantly lower heat pain thresholds and tolerances when stimulated on the volar surface of the left forearm. When examining visual analog ratings of intensity and unpleasantness to constant stimuli, a multivariate analysis of variance performed on these ratings indicated that there were significant main effects of level of stimulation and group. Individual analysis of variances at each temperature revealed significant differences between the groups in pain intensity and unpleasantness ratings at both noxious and innocuous temperatures. Multiple regression analyses indicated that greater pain catastrophizing and diagnosis of fibromyalgia were associated with decreased pain thresholds and tolerances in the entire sample, whereas, self‐report of depressive symptoms was associated with increased thresholds and tolerances. Self‐report of somatic symptoms was not associated with these measures. These findings indicate that persons with fibromyalgia display altered perception of both pressure and thermal stimulation, even at innocuous levels. They also suggest that catastrophic thoughts about pain may play a role in increased pain perception in this population.