Dean Willis

Inducible cyclooxygenase may have anti-inflammatory properties

Cyclooxygenase (COX) has two isoforms. Generally, COX 1 is constitutively expressed in most tissues, where it maintains physiological processes; inducible COX 2 is considered a pro-inflammatory enzyme and a chief target for the treatment of inflammatory diseases. Here we present evidence that COX 2 may have anti-inflammatory properties. In carrageenin-induced pleurisy in rats, the predominant cells at 2 hours are polymorphonuclear leucocytes, whereas mononuclear cells dominate from 24 hours until resolution at 48 hours. In this model, COX 2 protein expression peaked initially at 2 hours, associated with maximal prostaglandin E2 synthesis. However, at 48 hours there was a second increase in COX 2 expression, 350% greater than that at 2 hours. Paradoxically, this coincided with inflammatory resolution and was associated with minimal prostaglandin E2 synthesis. In contrast, levels of prostaglandin D2, and 15deoxyΔprostaglandin J2 were high at 2 hours, decreased as inflammation increased, but were increased again at 48 hours. The selective COX 2 inhibitor NS-398 and the dual COX 1/COX 2 inhibitor indomethacin inhibited inflammation at 2 hours but significantly exacerbated inflammation at 48 hours. This exacerbation was associated with reduced exudate prostaglandin D2 and 15deoxyΔprostaglandin J2 concentrations, and was reversed by replacement of these prostaglandins. Thus, COX 2 may be pro-inflammatory during the early phase of a carrageenin-induced pleurisy, dominated by polymorphonuclear leucocytes, but may aid resolution at the later, mononuclear cell-dominated phase by generating an alternative set of anti-inflammatory prostaglandins.

Cyclo-oxygenase and nitric oxide synthase isoforms in rat carrageenin-induced pleurisy.

1 The profiles of cyclo‐oxygenase (COX) and nitric oxide synthase (NOS) isoforms were determined in the rat carrageenin‐induced pleurisy model of acute inflammation. 2 The enzymes were assessed in peripheral blood leucocyte (PBL) cell pellets taken from untreated animals and at 2, 6 and 24 h after injection of the irritant in pleural exudate cell pellets and lung homogenates. 3 COX activity was assessed by the generation of prostacyclin (PGI2, measured as the stable metabolite, 6‐keto prostaglandin F1α) and prostaglandin E2 (PGE2). Western blot analysis and immunohistochemistry were also carried out. 4 NOS activity was based on the conversion of [3H]‐L‐arginine to [3H]‐L‐citrulline in the presence (total NOS activity) or absence of Ca2+ (inducible NOS; iNOS). 5 Peripheral blood leucocyte samples contained low levels of COX activity. In pleural exudate cell pellets, COX activity peaked at 2 to 6 h after injection of the carrageenin. At 24 h, COX activity was significantly reduced. 6 Western blot analysis demonstrated that the inducible isoform of COX (COX‐2), was the predominant enzyme at all time points. Low levels of COX‐2 were seen in PBLs. In pleural exudate cell pellets maximal COX‐2 protein levels were seen at 2h. 7 Immunohistochemistry confirmed the findings of Western blot studies. Approximately 10% of polymorphonuclear neutrophils (PMNs) in PBLs from untreated animals were immunopositive for COX‐2. In cell pellet smears from carrageenin‐induced pleurisy taken 2 h after injection of the irritant, PMNs were also the major source of COX‐2 immunoreactivity. A small proportion of macrophages and mesothelial cells were also immunolabelled for COX‐2. 8 Low levels of NOS activity were seen in PBLs. In pleural exudates NOS activity was maximum at 6h and greatly reduced by 24 h. This activity was solely attributable to iNOS. 9 The present results illustrated a similar profile of COX and NOS activity in the carrageenin‐induced pleurisy model of acute inflammation. It was demonstrated that COX‐2 and iNOS were the predominant isoforms of their respective enzymes.

Nitric Oxide Synthase Inhibitors Have Opposite Effects on Acute Inflammation Depending on Their Route of Administration

The bulk of published data has shown that NO is proinflammatory. However, there also exists the conflicting notion that NO may be protective during an inflammatory insult. In an attempt to resolve this issue, we have compared the effects on inflammation of a range of NO synthase (NOS) inhibitors given either directly to the site of the inflammatory lesion or systemically. It was found that in the carrageenin-induced pleurisy, a single intrapleural injection of the selective inducible NO inhibitors S-(2-aminoethyl) isothiourea (AE-ITU; 3 and 10 mg/kg) and N-(3-(aminomethyl)-benzyl) acetamidine (1400W; 10 mg/kg) or the selective endothelial cell NOS inhibitor l-N5(1-iminoethyl)-ornithine (10 mg/kg) not only exacerbated inflammation at the very early stages of the lesion (1–6 h), but also prevented inflammatory resolution. By contrast, administering NOS inhibitors systemically ameliorated the severity of inflammation throughout the reaction. To elucidate the mechanisms by which inhibition of NO synthesis locally worsened inflammation, we found an increase in histamine, cytokine-induced neutrophil chemoattractant, superoxide, and leukotriene B4 levels at the inflammatory site. In conclusion, this work shows that the local production of NO is protective by virtue of its ability to regulate the release of typical proinflammatory mediators and, importantly, that NOS inhibitors have differential anti-inflammatory effects depending on their route of administration.

THE ROLE OF VASCULAR ENDOTHELIAL GROWTH FACTOR IN A MURINE CHRONIC GRANULOMATOUS TISSUE AIR POUCH MODEL OF ANGIOGENESIS

Chronic granulomatous inflammation may be considered an angiogenic‐dependent process. Recently it has been demonstrated that vascular endothelial growth factor (VEGF) or vascular permeability factor is essential for tumour angiogenesis. Its role in inflammation‐mediated angiogenesis has yet to be determined. In this study, the murine chronic granulomatous air pouch model was used to investigate the role of VEGF in angiogenesis. Animals were treated twice weekly with 10 μg per animal of neutralizing antibody to rh VEGF and the vascularity and granuloma dry weight were assessed after 7 days. This resulted in significant suppression of both angiogenesis and granuloma dry weight. Western blot analysis demonstrated the presence of VEGF; the levels of protein paralleled the angiogenic response. These results demonstrate for the first time that VEGF may be an important regulator of angiogenesis in inflammation.

The peroxisome proliferator-activated receptor α activator, Wy14,643, is anti-inflammatory in vivo

The peroxisome proliferator-activated receptor system is exciting much interest as a novel point of therapeutic intervention in inflammation. Here, the effect of a peroxisome proliferatoractivated receptor α agonist, [4-chloro-6-(2,3-xylidine)-pyrimidinylthio]acetic acid (Wy14,643), was examined in arachidonic acid-induced murine ear inflammation. 3-[1-(4-Chlorobenzyl)-3-t-butyl-thio-5-isopropylindol-2-yl]-2,2-dimethylpropanoic acid (MK886, a 5-lipoxygenase inhibitor) and indomethacin (a cyclo-oxygenase inhibitor) were used as reference compounds. Wy14,643 dose dependently inhibited ear swelling and polymorphonuclear leukocyte influx, as did MK886, associated with reduced tissue leukotriene B4 but not prostaglandin E2 levels. Unlike MK886, Wy14,643 did not inhibit ex vivo leukotriene B4 production. However, Wy14,643, but not MK886, induced peroxisomal enzyme activity. Indomethacin was less effective, though tissue prostaglandin E2 but not leukotriene B4 levels were reduced. Again, unlike indomethacin, Wy14,643 did not reduce ex vivo prostaglandin E2 production. However, indomethacin did increase peroxisomal enzyme activity but to a lesser extent than Wy14,643. This study demonstrates that peroxisome proliferator-activated receptor α activation can inhibit arachidonic acid-induced inflammation in part by enhancing degradation of leukotriene B4.

Prostaglandin F2α produced by inducible cyclooxygenase may contribute to the resolution of inflammation

Cyclooxygenase-2 may play a role in resolution of carrageenan-induced pleurisy in rats by generating anti-inflammatory prostanoids. Here, we show exudate prostaglandin F2α concentrations rise during resolution of this model. These were reduced by the selective cyclooxygenase-2 inhibitor NS-398, which exacerbated inflammation. Concomitant treatment with NS-398 and the synthetic FP receptor agonist fluprostenol reversed this exacerbation. This suggests prostaglandin F2α produced by cyclooxygenase-2 contributes to resolution of this inflammatory reaction.

Marked Enhanced Efficacy of Cyclosporin When Combined with Hyaluronic Acid

SummaryUsing two models of chronic inflammation (both T-cell-mediated) in the Wistar rat, we have shown that, when given intravenously, the efficacy of cyclosporin can be markedly enhanced by combining it with sodium hyaluronate 7.5 mg/kg (HA; 500–800 kDa). When tested against avridine polyarthritis and cotton pellet granuloma formation, a marginally effective dose of cyclosporin 2.5 mg/kg could be transformed into a highly effective dose by combining it with HA 7.5 mg/kg. The latter combination appeared to mimic a highly efficacious dose of cyclosporin (10 mg/kg). Although the mechanism of this effect is as yet unclear, the potential clinical benefits are great.

Coronary vasoconstriction in vitro in the hearts of polyarthritic rats: effectiveness of in vivo treatment with the endothelin receptor antagonist SB 209670

Here we demonstrate that perfused hearts removed from polyarthritic rats develop a pronounced coronary vasoconstriction ex vivo. This vasoconstriction is almost entirely blocked by in vivo pretreatment of the rats with the endothelin receptor antagonist, SB 209670. Thus, inflammatory states may be associated with an increased activity of the endothelin system, leading to vascular dysfunction and vasoconstriction.

Relationship between apoptosis, angiogenesis and colon-26 tumour growth after oral nsaid treatment

Experimental and clinical studies have shown non-steroidal anti-inflammatory drugs (NSAIDs) as potential therapeutics in the treatment colorectal cancer. NSAIDs can reduce the relative risk of colorectal cancer in patients taking regular aspirin1. Also, sulindac can cause the regression of pre-existing colorectal cancers in patients with familial adenomatous polyposis (FAP)1.In vivoNSAIDs are protective in models of colonic carcinogenesisl and in the tumours of Apc(adenomatous polyposis coli)-mutatedMin mice2. However, the precise mechanism(s) for this chemopreventive action is unknown. Of the two isoenzymes of cyclooxygenase (COX, the classic target of NSAIDs), the inducible COX-2 enzyme expression is upregulated in 85% of colorectal cancer tissues, whilst COX-1 (the constitutive isoform) remains unchanged3. Experimentally, COX-2 overexpression has not only been shown to be an early event in the development of colorectal tumorigenesis4but also may have a protective role in preventing apoptosis5. This evidence therefore suggests selective targetting of COX-2 would be benefical as chemotherapeutic or chempreventive strategies. In this regard, selective COX-2 inhibition can inhibit polyp formation and size inApc Δ 716 knockout mice’, and reduce aberrant crypt foci (preneoplastic lesions) multiplicity, incidence and size during colonic carcinogenesis6. However, as a therapy for pre-existing disease (which comprises the majority of clinical presentations) less is known.

The role of the inducible enzymes cyclooxygenase-2, nitric oxide synthase and heme oxygenase in angiogenesis of inflammation

Angiogenesis, the formation of new blood vessels, is an essential part of the body’s physiology. In the non-pathological state, the process is largely quiescent and endothelial cell turnover may be measured in terms of years. However, it is an essential component of a variety of normal functions such as embryogenesis, normal tissue growth and the menstrual cycle. It also plays a role in the pathology of a variety of disease states and this led Judah Folkman, who many consider to be the “father of angiogenesis research”, to coin the term “angiogenesis-dependent disease” [1]. Some of the obvious examples which may be considered to fall into such a disease categorisation include neovascular glaucoma, hemangiomas and other tumors which need vascular support for their tissue expansion and metastatic activity. However, a number of other pathologies exist in which angiogenesis is a prominent feature; these include many of the chronic inflammatory diseases such as rheumatoid arthritis [2]. In these latter diseases, the neovasculature not only acts as a route for the increased nutrient supply required by the developing tissue, but also provides a greatly exaggerated area of activated endothelium which transmits proinflammatory signals, as well as receiving them, and allows the recruitment of large numbers of inflammatory leucocytes. The processes and cytokines involved in this proliferative capillary response have been recently reviewed [3, 4, 5]. The modulation of angiogenesis in inflammation therefore holds great therapeutic promise for the treatment of chronic inflammatory disease.