Protease phenotype of constitutive connective tissue and of induced mucosal mast cells in mice is regulated by the tissue.

Mouse mast cells (MCs) express a large number of serine proteases including tryptases, mouse mast cell protease (mMCP)-6 and -7; chymases, mMCP-1, -2, and -4; and an elastase, mMCP-5; along with carboxypeptidase-A3 (CPA3). In helminth-infected mouse intestine, distinct protease phenotypes are observed for connective tissue MCs (CTMCs) (mMCP-4(+)-7(+), and CPA3(+)) and mucosal MCs (MMCs) (mMCP-1(+) and 2(+)). To determine whether the protease phenotype was regulated by the tissue, we compared the phenotype of constitutive CTMCs and induced MMCs in trachea and large airways in antigen-sensitized unchallenged and challenged mice to MCs in skin and helminthic-infected intestine. We found that in the trachea, unlike in skin and intestine, CTMCs and MMCs both express all six serine proteases and CPA3 (mMCP-1(+), -2(+), 4(+)-7(+), CPA3(+)). This phenotype also holds for the lung CTMCs in the proximal bronchi, whereas the induced MMCs express only four proteases, mMCP-1, -2, -6, and -7. Thus, the T-cell-dependent induction of MMCs in trachea, large bronchi, and small intestine provides numbers but does not determine the protease phenotype. Furthermore, the CTMCs, which are constitutive, also show striking differences at these tissue sites, supporting the view that the differences in expression are tissue directed and not dependent on inflammation.

Epithelial and PGP9.5-immunoreactive cells of Malassez epithelium in the periodontal ligament of cats: a transmission electron microscopic study.

OBJECTIVE: The aim of the study was to investigate the ultrastructural features of Malassez epithelium (ME) containing protein gene product 9.5 (PGP9.5)-immunoreactive (IR) cells in the cat periodontal ligament (PDL). MATERIAL AND METHODS: Specimens from the teeth and tooth-supporting tissues of four mature cats of both sexes, 18 to 24 months of age, were used. The fixed jaws were decalcified in EDTA. Frozen sagittal sections 20 µm thick were immunostained for PGP9.5, and the ME, containing IR cells in the PDL, were evaluated under a transmission electron microscope. RESULTS: Several epithelial cells and PGP9.5 IR cells formed clusters and were enveloped by a basal lamina and separated from the surrounding connective tissue. A large nucleus and scanty cytoplasm were observed in most of the ME cells, which contained abundant keratin filaments and mitochondria. Caveolae-like structures and vesicles were found in the periphery of the ME. The small cytoplasmic processes of some of the epithelial cells extended toward the surrounding connective tissues. The cytoplasmic matrix of one type of cell comprising the ME showed immunoreactivity for anti-PGP9.5 antibody. The IR cell in the cell clusters was connected to adjacent epithelial cells and extended cytoplasmic processes toward the adjacent epithelial cells. The IR cell contained keratin filaments and abundant densely cored vesicles approximately 100-250 nm in diameter. CONCLUSIONS: The findings of the study suggest endocytotic capabilities of the epithelial cells and neuroendocrine functions of the IR cells. It is possible that the two different cell types react to extrinsic stimuli and interact with cells comprising the clusters and cords in the PDL. These ultrastructural evidences may imply functional heterogeneity of the ME in the PDL.

Potential for synthesis and degradation of extracellular matrix proteins by valve interstitial cells seeded onto collagen scaffolds.

Matrix remodeling, which involves proteolytic enzymes, such as the matrix metalloproteinases (MMPs), is of significant importance with respect to tissue engineering a heart valve construct. The ability of valve interstitial cells (ICs) to release these enzymes in biological scaffolds and to synthesize their own matrix has not been adequately studied, and this has important implications for tissue engineering. Cultured human aortic valve ICs were seeded onto a 3-dimensional type I collagen matrix for 28 days, whereby the presence of the remodeling enzymes, MMPs, were determined using immunohistochemistry, and detection of extracellular matrix (ECM) gene expression was performed using in situ hybridization. The collagenases, stromelysins, and membrane-type MMPs were expressed in 1%, 2%, and 5% collagen scaffolds after 28 days, whereas gelatinase expression was not observed. In situ hybridization revealed the presence of the ECM messenger ribonucleic acid (mRNA) in cells cultured in collagen scaffolds however, an increase in all three mRNAs was only detected in the 1% collagen scaffolds. The presence of collagenases, stromelysins, and membrane-type MMPs indicate that human valve ICs have the capacity to remodel type I collagen scaffold and that the genes necessary for synthesizing matrix have been turned on within the cells themselves. Scaffold composition also demonstrated differential effects onMMPexpression. These observations are of relevance with respect to the development of tissue-engineered heart valves.

Potential role of tissue transglutaminase in glaucoma filtering surgery.

PURPOSE: Scarring of the filtering bleb site is the main cause of failure in glaucoma filtration surgery. In the present study, the role of tissue transglutaminase (tTgase) in the accumulation of extracellular matrix (ECM) proteins in these scars was investigated. Transglutaminases are enzymes capable of cross-linking ECM proteins to proteolysis-resistant complexes. METHODS: Expression of tTgase, its reaction product epsilon-(gamma-glutamyl)-lysine, and fibronectin and their colocalization were investigated immunohistochemically in failed blebs and in an in vitro trabeculectomy model. Failed blebs were analyzed by RT-PCR for the presence of tTgase mRNA. Human Tenon fibroblasts (HTFs) were treated with transforming growth factor-beta2 (TGF-beta2). The effect was studied with immunohistochemistry, Northern blot analysis, and Western blot analysis. tTgase activity was assayed by incorporation of biotinylated cadaverine into fibronectin. RESULTS: Expression of tTgase and epsilon-(gamma-glutamyl)-lysine was present in all failed blebs. Staining was most prominent at the rim of the Tenon cyst. In the in vitro trabeculectomy model, tTgase and epsilon-(gamma-glutamyl)-lysine were barely present at the incision side of the flap but were perspicuously increased by TGF-beta2 treatment. Enzyme and its reaction product were colocalized with fibronectin. Cultured HTFs contained a basal level of tTgase mRNA. After treatment with TGF-beta2, expression and activity of tTgase significantly increased. CONCLUSIONS: The findings demonstrated that tTgase is present and functionally active in failed blebs. Expression and activity of tTgase appeared to be stimulated by TGF-beta2, a growth factor known to be increased in primary open angle glaucoma. Intervention at this pathway might open a new approach to prevent scarring after glaucoma filtration surgery.

A selective small molecule IkappaB Kinase beta inhibitor blocks nuclear factor kappaB-mediated inflammatory responses in human fibroblast-like synoviocytes, chondrocytes, and mast cells.

IkappaB kinase (IKK) beta is essential for inflammatory cytokine-induced activation of nuclear factor kappaB (NF-kappaB). NF-kappaB plays a pivotal role in the function of major cell types that contribute to the pathophysiological process of rheumatoid arthritis (RA). Here, we report the mechanism and the effect of the IKKbeta inhibitor N-(6-chloro-7-methoxy-9H-beta-carbolin-8-yl)-2-methylnicotinamide (ML120B), a beta-carboline derivative, on NF-kappaB signaling and gene activation in RA-relevant cell systems. ML120B is a potent, selective, reversible, and ATP-competitive inhibitor of IKKbeta with an IC50 of 60 nM when evaluated in an IkappaBalpha kinase complex assay. ML120B does not inhibit other IKK isoforms or a panel of other kinases. ML120B concentration-dependently inhibits tumor necrosis factor alpha (TNFalpha)-stimulated NF-kappaB signaling via inhibition of IkappaBalpha phosphorylation, degradation, and NF-kappaB translocation into the nucleus. For the first time, we have demonstrated that in human fibroblast-like synoviocytes, TNFalpha- or interleukin (IL)-1beta-induced monocyte chemoattractant protein-1 regulated on activation, normal T cell expressed and secreted and production is IKKbeta-dependent. In addition, for the first time, we have demonstrated that lipopolysaccharide- or peptidoglycan-induced cytokine production in human cord blood-derived mast cells is IKKbeta-dependent. In addition, in human chondrocytes, ML120B inhibited IL-1beta-induced matrix metalloproteinase production with an IC50 of approximately 1 microM. ML120B also blocked IL-1beta-induced prostaglandin E2 production. In summary, ML120B blocked numerous NF-kappaB-regulated cell responses that are involved in inflammation and destructive processes in the RA joint. Our findings support the evaluation of IKKbeta inhibitors as anti-inflammatory agents for the treatment of RA.

Mitomycin-C induces the apoptosis of human Tenon's capsule fibroblast by activation of c-Jun N-terminal kinase 1 and caspase-3 protease.

PURPOSE: To investigate whether mitochondrial dysfunction and mitogen-activated protein kinase family proteins are implicated in apoptotic signaling of human Tenon's capsule fibroblasts (HTCFs) by mitomycin-C. METHODS: Apoptosis was determined by Hoechst nuclei staining, agarose gel electrophoresis, and flow cytometry in HTCFs treated with 0.4 mg/mL mitomycin-C for 5 minutes. Enzymatic digestion of florigenic biosubstrate assessed the catalytic activity of caspase proteases, including caspase-3, caspase-8, and caspase-9. Phosphotransferase activity of c-Jun N-terminal kinase (JNK) 1 was measured by in vitro immune complex kinase assay using c-Jun(1-79) protein as a substrate. Mitochondrial membrane potential transition (MPT) was measured by flow cytometric analysis of JC-1 staining. RESULTS: Mitomycin-C (0.4 mg/mL) induced the apoptosis of HTCFs, which was characterized as nucleic acid and genomic DNA fragmentation, chromatin condensation, and sub-G(0)/G(1) fraction of cell cycle increase. The catalytic activity of caspase-3 and caspase-9 was significantly increased and was accompanied by cytosolic release of cytochrome c and MPT in response to mitomycin-C. Treatment with mitomycin-C resulted in the increased expression of Fas, FasL, Bad, and phosphorylated p53 and a decreased level of phosphorylated AKT. Treatment with mitomycin-C also increased the phosphotransferase activity and tyrosine phosphorylation of JNK1, whose inhibitor significantly suppressed the cytotoxicity of mitomycin-C. CONCLUSIONS: Mitomycin-C induced the apoptosis of HTCFs through the activation of intrinsic and extrinsic caspase cascades with mitochondrial dysfunction. It also activated Fas-mediated apoptotic signaling of fibroblasts. Furthermore, the activation of JNK1 played a major role in the cytotoxicity of mitomycin-C.

Calcification of human valve interstitial cells is dependent on alkaline phosphatase activity.

BACKGROUND AND AIM OF THE STUDY: The calcification of heart valves is associated with valve degeneration and failure, but the mechanisms involved are poorly understood. The presence of lamellar bone has been demonstrated in calcified aortic valves. Since osseous calcification is closely associated with alkaline phosphatase (ALP) activity, it was hypothesized that ALP activity might be implicated in the calcification of isolated leaflet interstitial cells (ICs). METHODS: Human valve leaflet ICs were isolated from transplant-explanted hearts at the time of transplantation (n = 12). RESULTS: Isolated leaflet ICs expressed the fibroblast-specific antigen (100% of cells) and smooth muscle (SM) alpha-actin (70-80% of cells), but osteoblastic markers were not expressed. Cultured ICs did not calcify spontaneously, however when the growth medium was supplemented with beta-glycerophosphate (an organic phosphate) it induced the formation of calcified nodules that expressed osteonectin and ALP, but not SM alpha-actin. Beta-glycerophosphate-induced calcification of ICs showed a time-dependent effect on the calcium content of treated cells over a 14-day period. ALP activity was considerably increased in beta-glycerophosphate-treated ICs, and this correlated with the calcium content (r = 0.5: p = 0.01). Levamisol (an ALP inhibitor) inhibited the beta-glycerophosphate-induced calcification process, as well as the expression of osteoblastic differentiation markers. CONCLUSION: Isolated and cultured leaflet ICs did not calcify spontaneously, though organic phosphate induced the formation of calcified nodules that expressed osteoblastic markers. The calcification of isolated ICs was seen to be dependent on ALP activity.

Stimulation of pro-MMP-2 production and activation by native form of extracellular type I collagen in cultured hepatic stellate cells.

Cultured hepatic stellate cells (HSCs) are known to change their morphology and function with respect to the production of extracellular matrices (ECMs) and matrix metalloproteinases (MMPs) in response to ECM components. We examined the regulatory role of the native form of type I collagen fibrils in pro-MMP-2 production and activation in cultured HSCs. Gelatin zymography of the conditioned media revealed that pro- and active form of MMP-2 was increased in the HSCs cultured on type I collagen gel but not on type I collagen-coated surface, gelatin-coated surface, type IV collagen-coated surface, or Matrigel, suggesting the importance of the native form of type I collagen fibrils in pro-MMP-2 production and activation. The induction of active MMP-2 by extracellular type I collagen was suppressed by the blocking antibody against integrin beta1 subunits, indicating the involvement of integrin signaling in pro-MMP-2 activation. RT-PCR analysis indicated that MMP-2, membrane type-1 MMP (MT1-MMP) and tissue inhibitor of metalloproteinase-2 (TIMP-2) mRNA levels were elevated in HSCs cultured on type I collagen gel. The increased MT1-MMP proteins were localized on the cell surface of HSCs cultured on type I collagen gel. In contrast to the expression of MMP-2, HSCs showed a great decline in MMP-13 expression in HSCs cultured on type I collagen gel. These results indicate that the native fibrillar (polymerized) but not monomeric form of type I collagen induced pro-MMP-2 production and activation through MT1-MMP and TIMP-2 in cultured HSCs, suggesting an important role of HSCs in ECM remodeling in the hepatic perisinusoidal spaces.

The reactive adventitia: fibroblast oxidase in vascular function.

The vascular adventitia is activated in a variety of cardiovascular disease states and has recently been shown to be a barrier to nitric oxide bioactivity. Vascular fibroblasts produce substantial amounts of NAD(P)H oxidase-derived reactive oxygen species (ROS) that appear to be involved in fibroblast proliferation, connective tissue deposition, and perhaps vascular tone. However, the physiological and pathophysiological roles of the adventitia have not been extensively studied, possibly because of its location in large blood vessels remote from the vascular endothelium. In recent years, substantial information has been gathered on pathways leading to oxidase activation in smooth muscle cells and fibroblasts and the downstream signaling pathways leading to hypertrophy and proliferation. A clearer understanding of the molecular mechanisms involved will likely lead to therapeutic strategies aimed at preventing vascular dysfunction in diseases such as atherosclerosis, in which these pathways are activated.

Oxidative stress induces heme oxygenase-1 immunoreactivity in Müller cells of mouse retina in organ culture.

PURPOSE: Heme oxygenase (HO)-1 immunoreactivity (IR) was examined in normal untreated retina and in retinal explants after in vitro treatment with stress agents. METHODS: Enucleated eyes from young adult C3H mice were immediately fixed and cryosectioned and the retina sections processed for immunocytochemistry with antibodies against HO-1 and glial fibrillary acidic protein (GFAP). From other eyes retinas were isolated and maintained in organ culture, either untreated for 4 days maximum or for 21 hours during which the explants were treated the first 3 hours with selected doses of sodium arsenate or hydrogen peroxide. Thereafter, the explants were processed identically with the normal tissue. RESULTS: In the normal retina, HO-1 and GFAP IR was very low. The culturing itself resulted in an increase in both HO-1 and GFAP immunolabeling in Müller cells of explanted retinas. Both sodium arsenate and hydrogen peroxide further induced strong HO-1 IR in Müller cells but not in other retinal cells. In contrast to HO-1, GFAP staining in Müller cells was not altered as a result of treatment, either by sodium arsenate or hydrogen peroxide at any concentration used. CONCLUSIONS: The results show for the first time that HO-1 can be induced in the retina in vitro by conditions of oxidative stress and that enzyme expression is confined exclusively to Müller cells.

Cyclooxygenase-2 is upregulated in inflamed gingival tissues.

BACKGROUND: Increased release of prostaglandins (PG) within periodontal tissues is considered to play a pathogenetic role during periodontal disease progression. The rate-limiting step in the formation of PG from arachidonic acid is catalyzed by cyclooxygenase (COX). Currently there are 2 known isoforms of the enzyme. COX-1 is constitutively expressed in various tissues whereas COX-2 is an inducible enzyme believed to be responsible for PG synthesis at sites of inflammation. The purpose of this study was to compare COX-2 expression in inflamed and healthy human gingiva and further explore some of the pathogenetic mechanisms which may lead to elevated COX-2 expression in vivo. METHODS: Thirty-two gingival biopsies were obtained during routine oral surgical procedures and were processed histologically using hematoxylin and eosin to determine the degree of inflammation. Of these biopsies, 7 with low and 7 with high histological levels of inflammation were further processed immunohistochemically in order to assess the levels of COX-2 expression in situ. To explore some potential mechanisms of COX-2 upregulation, gingival connective tissue primary cell cultures were established and challenged with periodontal bacteria or proinflammatory cytokines in vitro. The levels of COX-2 expression were analyzed by Western blot of cell lysates. COX-2 activity was assessed by quantifying prostaglandin E2 (PGE2) levels in culture supernatants by competitive EIA. RESULTS: We have shown by immunohistochemistry that COX-2 expression was significantly higher (P < 0.01) in tissues with higher levels of inflammatory infiltrates. Expression of COX-2 was detected in gingival epithelium, endothelial cells as well as cells with fibroblast morphology. In vitro studies indicated that gingival fibroblasts (GF) did not express COX-2 constitutively. However, when these cells were challenged with interleukin (IL)-1 beta or bacterial cells (A. actinomycetemcomitans JP2 or B. forsythus ATCC 43037), COX-2 expression as well as COX-2 activity were upregulated. COX-2 expression was upregulated as early as 2 hours post IL-1 beta challenge and was accompanied by a sustained PGE2 release in the culture supernatants. Cyclosporin A (CsA) did not inhibit COX-2 expression induced by bacterial challenge. In contrast, NS-398, a selective inhibitor of COX-2 activity, almost completely abolished PGE2 synthesis by these cells in response to bacterial or cytokine challenge. CONCLUSIONS: We conclude that COX-2 expression is significantly upregulated in inflamed periodontal tissues. Both inflammatory cytokines such as IL-1 beta and bacterial constituents may be responsible for the enhanced COX-2 expression and PGE2 synthesis in vivo.

Enzyme-histochemical detection of a chymase-like proteinase within bovine mucosal and connective tissue mast cells.

The presence of chymase-like proteinase in bovine mast cells was investigated by an enzyme-histochemical technique (naphthol-AS-D-chloroacetate as substrate) in normal skin, primary bronchus, lung and duodenum. The counts and distribution of chymase-positive and toluidine blue-positive mast cells were compared by means of successive staining. Mast cells with chymase-like activity were detected in all areas, but their proportion was greater in connective than mucosal tissues, with the exception of the skin. These results contrast with those obtained in rodents, in which chymase-like proteinases are detected in all tissues and also in all mast cells. Bovine mast cells are closer to those of human beings, in which chymase-containing mast cells predominate in connective tissues, including skin. The results suggest that more than one chymase subset is present, at least in duodenum. The possible occurrence of dual-specific chymase mast cells, as in other ruminants, is discussed.

Collagenolytic activity of cathepsin K is specifically modulated by cartilage-resident chondroitin sulfates.

Cathepsin K is the predominant cysteine protease in osteoclast-mediated bone remodeling, and the protease is thought to be involved in the pathogenesis of diseases with excessive bone and cartilage resorption. Osteoclastic matrix degradation occurs in the extracellular resorption lacuna and upon phagocytosis within the cell's lysosomal-endosomal compartment. Since glycosaminoglycans (GAGs) are abundant in extracellular matrixes of cartilage and growing bone, we have analyzed the effect of GAGs on the activity of bone and cartilage-resident cathepsins K and L and MMP-1. GAGs, in particular chondroitin sulfates, specifically and selectively increased the stability of cathepsin K but had no effect on cathepsin L and MMP-1. GAGs strongly enhanced the stability and, to a lesser extent, the catalytic activity of cathepsin K. To combine the activity and stability parameters, we defined a novel kinetic term, named cumulative activity (CA), which reflects the total substrate turnover during the life span of the enzyme. In the presence of chondroitin-4-sulfate (C-4S), the CA value increased 200-fold for cathepsin K but only 25-fold with chondroitin-6-sulfate (C-6S). C-4S dramatically increased the hydrolysis of soluble as well insoluble type I and II collagens, whereas the effects of C-6S and hyaluronic acid were less pronounced. C-4S acts in a concentration-dependent manner but reaches saturation at approximately 0.1%, a concentration similar to that found in the synovial fluid of arthritis patients. C-4S increased the cathepsin K-mediated release of hydroxyproline from insoluble type I collagen 10-fold but had only a less than 2-fold enhancing effect on the hydrolysis of intact cartilage. The relatively small increase in the hydrolysis of cartilage by C-4S was attributed to the endogenous chondroitin sulfate content present in the cartilage. Although C-4S increased the pH stability at neutral pH, a significant increase in the collagenolytic activity of cathepsin K at this pH was not observed, thus suggesting that the unique collagenolytic activity of cathepsin K at acidic pH is mechanistically determined and not by the enzyme's instability at neutral pH. The selective and significant stabilization and activation of cathepsin K activity by C-4S may provide a rationale for a novel mechanism to regulate the enzyme's activity during bone growth and aging, two processes known for significant changes in the GAG content.

A recombinant human TGF-beta1 fusion protein with collagen-binding domain promotes migration, growth, and differentiation of bone marrow mesenchymal cells.

A continuous source of osteoblasts for normal bone maintenance, as well as remodeling and regeneration during fracture repair, is ensured by the mesenchymal osteoprogenitor stem cells of the bone marrow (BM). The differentiation and maturation of osteoprogenitor cells into osteoblasts are thought to be modulated by transforming growth factors-beta (TGF-beta1 and TGF-beta2) and TGF-beta-related bone morphogenetic proteins (BMPs). To define the responses of mesenchymal osteoprogenitor stem cells to several growth factors (GFs), we cultured Fischer 344 rat BM cells in a collagen gel medium containing 0.5% fetal bovine serum for prolonged periods of time. Under these conditions, survival of BM mesenchymal stem cells was dependent on the addition of GFs. Recombinant hTGF-beta1-F2, a fusion protein engineered to contain an auxiliary collagen binding domain, demonstrated the ability to support survival colony formation and growth of the surviving cells, whereas commercial hTGF-beta1 did not. Initially, cells were selected from a whole BM cell population and captured inside a collagen network, on the basis of their survival response to added exogenous GFs. After the 10-day selection period, the surviving cells in the rhTGF-beta1-F2 test groups proliferated rapidly in response to serum factors (10% FBS), and maximal DNA synthesis levels were observed. Upon the addition of osteoinductive factors, osteogenic differentiation in vitro was evaluated by the induction of alkaline phosphatase (ALP) expression, the production of osteocalcin (OC), and the formation of mineralized matrix. Concomitant with a down-regulation of cell proliferation, osteoinduction is marked by increased ALP expression and the formation of colonies that are competent for mineralization. During the induction period, when cells organize into nodules and mineralize, the expression of OC was significantly elevated along with the onset of extracellular matrix mineralization. Differentiation of BM mesenchymal stem cells into putative bone cells as shown by increased ALP, OC synthesis, and in vitro mineralization required the presence of specific GFs, as well as dexamethasone (dex) and beta-glycerophosphate (beta-GP). Although rhTGF-beta1-F2-selected cells exhibited the capacity to mineralize, maximal ALP activity and OC synthesis were observed in the presence of rhBMPs. We further report that a novel rhTGF-beta1-F2 fusion protein, containing a von Willebrand's factor-derived collagen binding domain combined with a type I collage matrix, is able to capture, amplify, and stimulate the differentiation of a population of cells present in rat BM. When these cells are subsequently implanted in inactivated demineralized bone matrix (iDBM) and/or diffusion chambers into older rats they are able to produce bone and cartilage. The population of progenitor cells captured by rhTGF-beta1-F2 is distinct from the committed progenitor cells captured by rhBMPs, which exhibit a considerably more differentiated phenotype.

Effect of diclofenac sodium and dexamethasone on cultured human Tenon's capsule fibroblasts.

BACKGROUND AND OBJECTIVE: To investigate the effect of diclofenac sodium and dexamethasone on cultured human Tenon's capsule fibroblasts. MATERIALS AND METHODS: Two experiments were conducted. In the first experiment, fibroblasts were treated with either diclofenac sodium or dexamethasone at different concentrations, and the cell growth was quantified by using Coulter counter and hexosaminidase methods at 1, 3, 5, and 7 days after adding the drugs. In the second experiment, the cells were treated with each drug for 24 hours and then the cultures were switched to a drug-free medium. The cell growth was quantified at day 7 after removing the drugs from the medium. RESULTS: In the first experiment, inhibition of fibroblast growth in a dose-dependent manner was observed from days 1 to 7 in the cultures treated with each drug. The inhibitory was more pronounced in the diclofenac treated cultures. The typical spindle-shaped fibroblasts treated with higher concentrations of the drugs became spherical cells. In the second experiment, inhibition was not observed when the cultures were switched to a drug-free medium. The spherical cells recovered to spindle-shaped cells and proliferated as normal cells. CONCLUSION: Our results have shown that diclofenac sodium and dexamethasone can significantly inhibit human Tenon's capsule fibroblast growth in a cell culture model. The inhibitory effect was not observed when the cultures were switched after 24 hours to a drug-free culture medium.

Induction of cyclooxygenase-2 by secretory phospholipases A2 in nerve growth factor-stimulated rat serosal mast cells is facilitated by interaction with fibroblasts and mediated by a mechanism independent of their enzymatic functions.

Mast cells exhibit a biphasic (immediate and delayed) eicosanoid-biosynthetic response after stimulation with particular cytokines or Fc epsilonRI (high affinity receptor for IgE) cross-linking. Treatment of rat serosal connective tissue mast cells (CTMC) with nerve growth factor (NGF) induced only the delayed phase of PGD2 generation that depended on inducible cyclooxygenase-2 (COX-2), but not constitutive COX-1, even though the subcellular distributions of these isoforms were similar. Experiments using several phospholipase A2 (PLA2) isozyme-specific probes and inhibitors suggested that both constitutive cytosolic PLA2 and inducible type IIA secretory PLA2 (sPLA2) are involved in NGF-initiated, COX-2-dependent, delayed PGD2 generation in rat CTMC. A type IIA sPLA2 inhibitor, but neither cytosolic PLA2 nor COX inhibitors, reduced, while adding exogenous type IIA sPLA2 augmented, NGF-induced COX-2 expression and its attendant PGD2 generation, indicating that the sPLA2-mediated increase in delayed PGD2 generation was attributable mainly to enhanced COX-2 expression. Type IIA sPLA2 and its close relative type V sPLA2 associated with fibroblastic cell surfaces increased NGF-induced COX-2 expression more efficiently than the soluble enzymes, revealing a particular juxtacrine sPLA2 presentation route. Surprisingly, catalytically inactive type IIA sPLA2 mutants, which were incapable of promoting arachidonic acid release from cytokine-primed cells, retained the ability to enhance COX-2 expression in CTMC, indicating that the COX-2-inducing activities of sPLA2 are independent of their catalytic functions.