Amelioration of TNBS-induced colon inflammation in rats by phospholipase A2 inhibitor

M. Krimsky,1 S. Yedgar,1 L. Aptekar,2 O. Schwob,1 G. Goshen,3 A. Gruzman,4 S. Sasson,4 and M. Ligumsky2

1Department of Biochemistry, Hadassah Medical School, 2Gastroenterology Unit, Hadassah University Hospital, 3Department of Oral Biology, School of Dental Medicine, and 4Department of Pharmacology, School of Pharmacy, Hebrew University Faculty of Medicine, Jerusalem, Israel 91120

Submitted 28 October 2002 ; accepted in final form 23 April 2003


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
The pathophysiology of inflammatory bowel disease (IBD) involves the production of diverse lipid mediators, namely eicosanoids, lysophospholipids, and platelet-activating factor, in which phospholipase A2 (PLA2) is the key enzyme. Accordingly, it has been postulated that control of lipid mediator production by inhibition of PLA2 would be useful for the treatment of IBD. This hypothesis was tested in the present study by examining the therapeutic effect of a novel extracellular PLA2 inhibitor (ExPLI), composed of carboxymethylcellulose-linked phosphatidylethanolamine (CMPE), on trinitrobenzenesulfonic acid-induced colitis. Intraperitoneal administration of CMPE suppressed the colitis as measured by mortality rate, intestinal permeability, plasma PLA2 activity, intestinal myeloperoxidase activity, and histological morphometry. Current therapeutic approaches for inflammatory conditions focus on the selective control of a lipid mediator(s) (e.g., prostaglandins or leukotrienes). The present study supports the concept that inclusive control of lipid mediator production by PLA2 inhibition is a plausible approach to the treatment of colitis and introduces the ExPLIs as a prototype of a novel NSAID for the treatment of intestinal inflammation.

inflammatory bowel disease; colitis; nonsteroidal anti-inflammatory drugs


INFLAMMATORY BOWEL DISEASE (IBD) is a chronic disorder of the intestine, relating mainly to ulcerative colitis and Crohn's disease (43), associated with abdominal pain, diarrhea, fever, weight loss, anemia, arthritic pain, and impairment of liver function. Although the etiology of IBD is not known, its pathophysiology is postulated to have two stages: an insult triggering initial tissue damage, followed by an amplification stage, which propagates the tissue destruction and the duration of the disease. It is assumed that in the amplification stage, inflammatory cells migrate to the injured site and produce diverse proinflammatory mediators, mainly eicosanoids [prostaglandins (PGs), thromboxanes (TXs), leukotrienes (LTs)] and cytokines (34, 40, 43).

Because different eicosanoids are potent mediators of inflammatory processes, the inhibition of their production has long been proposed and tested for therapeutic purposes. The eicosanoids include two main families of arachidonic acid (AA) metabolites derived through the cyclooxygenase pathways (COX-1 and COX-2), producing PGs and TXs, or the lipoxygenase (LOX) pathways, producing LTs and hydroxyeicosatetraeinoics (HETEs). In addition, eicosanoid production in inflammatory processes is linked to cytokine production, although the cause-and-effect relationship and interaction between these two groups of inflammatory mediators is not clear (1, 5, 7, 40, 43). Accordingly, the control of eicosanoid production has been a major target in the treatment of inflammatory processes. The prevalent approach has been aimed at selective inhibition of the production of a specific central eicosanoid or a related enzymatic pathway (e.g., COX-2 inhibition). However, these compounds play a complex role in the pathophysiology of inflammatory conditions: each of these pathways may produce agonists and antagonists of the same biological process. Moreover, similar effects may be exerted by products of COX-1, COX-2, and LOX (25, 39). Thus it often happens that selective blocking of one pathway diverts the AA pool to the alternative pathway(s) (17, 25), leading to exacerbation of the pathological state (3, 17, 26). One such situation is IBD, in which products of both the cyclooxygenase pathway (PGE2, PGF2{alpha}, PGD2, thromboxane B2) and the lipoxygenase pathway (leukotriene B4, 5-HETE, 12-HETE) have been reported to be involved in the acceleration of the inflammatory process (13, 40, 43). In addition, COX-1 and COX-2 play a complex role in gastrointestinal inflammation, and disparate views have been expressed as to their part in its pathophysiology (13, 40, 48).

On these grounds, the "dual-pathway inhibition" has been proposed, aiming at suppressing the activation of eicosanoid production by controlling the release of their precursor, AA (17, 19, 26), i.e., inhibiting phospholipase A2 (PLA2) activity.

PLA2 hydrolyzes membrane phospholipids to produce free fatty acids and lysophospholipids and thus initiates the production of the different families of inflammatory lipid mediators: the AA-derived eicosanoids, different lysophospholipids, and platelet-activating factor (PAF) (20, 33). Diverse lipid mediators, specifically derived by the COX and lipoxygenase pathways, as well as PAF, have been found to be strong mediators of intestinal inflammation (40). In addition, PLA2, which is secreted excessively into body fluids in inflamed tissues, synergizes with reactive oxygen species to induce cell lysis in host tissues (12, 15), and, ultimately, the actual lysis of the cell membrane lipids is done directly by the extracellular PLA2 (12, 39). In addition, PLA2 activity is linked to cytokine production by inflammatory cells, either directly or via its effect on eicosanoid production, although the interrelation between them is not yet clear (1, 5, 31). Activation of PLA2, particularly the secretory enzyme (sPLA2), has been implicated in different types of intestinal inflammation in humans (7, 18, 29, 30, 41) and in animal models (14, 21). Altogether, PLA2 has long been considered a pivotal enzyme in inflammatory and allergic processes (21, 33), and inhibition of its activity might exert multiple effects in amelioration of related diseases (21, 26, 36).

Accordingly, steroids, which are often used for treating IBD, are assumed to suppress PLA2 activity through lipocortin synthesis (27). In addition, it has been shown that sulfasalazine, used to treat IBD, inhibits sPLA2 release (32). Inhibitors of PLA2, such as mepacrine (41), quinacrine, and p-bromophenacylbromide (PBPB) (14, 21, 29), have been shown to suppress intestinal inflammation in animal models (14, 21) or in biopsies of patients' intestine (29, 42).

Because of the central role of PLA2 in inflammatory processes, inhibitors of its activity have been sought for treatment of related diseases (8, 26, 38, 39). However, since inhibitors that penetrate the cell (e.g., mepacrine, quinacrine, PBPB) might impair the cell metabolism and viability, it has long been suggested that the appropriate treatment requires an inhibitor of PLA2 activity at the cell membrane that is not internalized by the cell (8). Extracellular PLA2 inhibitors (ExPLIs) that fulfill these requirements have been designed and synthesized in the lab of S. Yedgar by binding PLA2-inhibiting molecules to polymeric carriers in a way that enables the inhibiting moiety to control the cell membrane PLA2 activity, but its internalization is prevented by the polymeric carrier (11, 47). The ExPLIs have been found effective in protecting the membrane from different types of exogenous PLA2, including porcine pancreatic and human recombinant synovial fluid PLA2 (11), protecting cells from activation of endogenous PLA2 and concomitant cell damage induced by proinflammatory agents (16, 45), inhibiting LPS-induced activation of endogenous PLA2 in macrophages (2), suppressing IFN-{gamma}-induced activation of endothelial cells (44), suppressing LPS-induced chemokine production by endothelial cells (5), as well as ameliorating bleomycin-induced lung injury in hamsters (10) and endotoxin-induced sepsis in rats (4).

On these grounds, the present study was undertaken to test the prospect of the multiple pathway control of lipid mediator production in the treatment of IBD by testing ExPLIs' effect on experimental intestinal inflammation. For this purpose, we have examined the effect of the ExPLI composed of N-derivatized phosphatidylethanolamine linked to carboxymethylcellulose (CMPE) (11) on colitis induced in rats by trinitrobenzenesulfonic acid (TNBS). Human IBD is unique, and animal models do not fully simulate its pathophysiology and clinical course. Despite these drawbacks, this model is widely accepted for the study of colon inflammation and its treatment. In the present study, the treatment with ExPLIs was found considerably effective in amelioration of TNBS-induced colitis, as measured by mortality, intestinal permeability and histology, and biochemical markers.


    MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
TNBS-induced colitis. Following a conventional method (21, 22, 35), TNBS (Sigma, St. Louis, MO) was administered rectally (25 mg in 1 ml of 50% ethanol) to Hebrew University Sabra rats (200-250 g) after 24 h of fasting, and the course of the disease was monitored for 2 days, according to our preliminary experiments and previous reports (25). On the basis of previous experience, in the ExPLI-treated group, the rats were treated intraperitoneally with 10 mg/100 g body wt CMPE (hereafter CMPE-treated) in 1 ml saline (to obtain ~10 µM in body fluid) at 18 and 0.5 h before as well as 3, 18, and 36 h after TNBS administration to maintain a significant level of CMPE during the experiment course. The untreated rats (hereafter TNBS rats) received 1 ml of the vehicle (saline) intraperitoneally at the same time points.

Histological morphometry of intestinal damage. The rats that survived the disease 48 h after induction were terminated under ether anesthesia, and 10-cm segments of the distal colon were evaluated for macroscopic histological damage and then dissected longitudinally into two parts. One part was immediately fixed in 10% formalin and embedded in paraffin following dehydration with ethanol; the other part was used for determination of myeloperoxidase (MPO) activity.

Macroscopic scoring of intestinal damage, from 0 (no damage) to 5 (maximal damage) was assessed by a naked-eye examination of areas of mucosal discoloration, erosion, exudation, ulceration, bowel wall thickening, and percentage of damaged area.

For histological morphometry, serial sections 5-µm thick were cut perpendicularly to the intestinal wall and stained with hematoxylin and eosin. The ulcerative area was determined on histological slides by using light microscopic image processes by a CUE-3 image analysis system (Galai, Migdal Haemek, Israel). As previously described (6), the CUE-3 system comprises high-resolution color charge-coupled device cameras (M-852; Sony) and a monitor (Trinitron; Sony). Image analysis was carried out in a predefined image window (with a surface of 0.3 mm2 and by color thresholding representing the "actual objects" in a given window). For each rat, histology slides were taken from five loci along the intestine dissection (the same loci were taken from all dissections). The measurements were quantified from 20-30 microscopic fields in 5 histology slides for each rat.

Intestinal permeability. Intestinal permeability was evaluated by determining the level of inulin-fluorescein (InFl, Sigma) in the plasma following its rectal administration (25). In a preceding study (25), we found that, although InFl does not permeate the intestine of normal rats, it is readily adsorbed in the colitic rats. That study showed that, in rats with TNBS-induced colitis, the intestinal permeability, as measured by InFl in plasma, reaches its peak at 12 h after administration of TNBS. Accordingly, in the present study, InFl (4 mg in 0.2 ml saline) was given rectally to both TNBS and CMPE-treated groups 12 h after administration of TNBS, and a blood sample (0.5 ml) was taken 2 h later from the tail vein of the ether-anesthetized rats (25). For determination of blood level of InFl, the plasma was separated by centrifugation and the fluorescence intensity was determined at 488 nm (excitation) and 517 nm (emission) as previously described (25).

PLA2 activity secreted into blood serum. Blood samples (0.3 ml) were drawn from the rats by cardiac puncture under anesthesia at several time points. Before this experiment, we tested the possible effect of this procedure on plasma PLA2 level by performing the same repeated cardiac puncture on normal (untreated) rats and found that this procedure did not affect the plasma PLA2 activity. To avoid frequent anesthesia, in the present experiment, blood samples were drawn from each rat only three times. Accordingly, for each treatment (TNBS and TNBS + CMPE), we used two sets of five rats: one set of rats was subjected to blood sampling at 1, 6, and 12 h and the other set at 2, 6, and 24 h after TNBS administration (see Fig. 3). A slight difference was observed between the two sets at 6 h, and to obtain a continuous curve for all time points, the data for the two sets were normalized (by adjustment of the 6-h values of the 2 sets).



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Fig. 3. Effect of CMPE on phospholipase A2 (PLA2) level in serum of rats with TNBS-induced colitis. Blood from rats with TNBS-induced colitis, either untreated (black bars) or treated with CMPE, as described in MATERIALS AND METHODS, was drawn at the indicated time after induction of the disease with TNBS. Serum was separated by centrifugation, and its PLA2 activity was determined as described in MATERIALS AND METHODS. Each datum is a mean ± SE for 5 rats (3 replications for each serum sample). The statistical significance between the 2 sets of data, as determined by {chi}-square test for combined probabilities, is P = 0.0114.

 

The plasma of the drawn blood samples was separated by centrifugation, and its PLA2 activity was determined by the hydrolysis of phospholipid membranes (liposomes), as previously described (11). In brief, for preparation of lipid vesicles, 1-oleoyl-lysophosphatidylcholine and dipalmitoylphosphatidylcholine (DPPC) containing 2-[14C]DPPC (specific radioactivity = 1 nCi/nmol) dissolved in chloroform were mixed at a molar ratio of 2:1, respectively, dried under nitrogen, and suspended in Ca2+-free Tris buffer (100 mM, pH 8.0) by cyclomixer to obtain total lipid concentration of 1.0 mM. For determination of serum PLA2 activity, 25 µl of serum were mixed with 50 µl lipid vesicles and supplemented with Tris buffer containing 1 mM Ca2+ and 2% fatty acid-free bovine serum albumin to a final volume of 200 µl and were incubated at 37°C for the desired time. The reaction was terminated by the addition of organic solvent (Dole) to form two phases (37). The organic phase was collected, and its radioactivity was determined in a {beta}-scintillation counter. The activity in blood before induction of disease was taken as background and subtracted from the values obtained after disease induction. It has previously been shown, using thin-layer chromatography, that with the use of this method, the organic phase contains all of the free fatty acid released but the amount of substrate is insignificant (23, 46). This was reconfirmed by us by using thin-layer chromatography before employing this method in the present study. As noted above, the amount of CMPE given to the treated rats was aimed at obtaining a concentration of ~10 µM in body fluid. Accordingly, to rule out the possibility that the change in plasma PLA2 activity in CMPE-treated rats is due to its presence in plasma, the same concentration of CMPE was added to blood samples drawn from the TNBS rats (untreated with CMPE) before the PLA2 activity was assayed.

The substrate used here (DPPC) is not hydrolyzed by cytosolic PLA2 (cPLA2), which is specific to AA-carrying phospholipid (2), and its hydrolysis thus represents the activity of sPLA2 secreted to blood plasma.

MPO activity. Colon mucosa was homogenized in PBS (pH 7.4), and MPO activity was determined spectroscopically by o-dianisidine/H2O2 reaction (9, 14). Because of the high mortality rate among the TNBS (untreated) rats, MPO determinations were limited to the rats that survived the course of the experiment. This introduced a methodological problem, because it is likely that the rats that died had the most severe damage but these could not be included in the comparison between treated and untreated animals. This drawback was taken into consideration when analyzing the data of MPO activity and InFl permeation, as described in RESULTS.

All of the procedures applied to the rats were approved by the Hebrew University Institutional Animal Care and Use Committee (IACUC; permit OPRR-A01-5011).

Statistical analysis. The results are expressed as means ± SE. For Figs. 1 and 2, the difference between the TNBS and CMPE-treated groups was examined for significance by the Mann-Whitney U-test. For Fig. 3, the difference was examined for significance by the Mann-Whitney {chi}-square test for combined probabilities (which compare the two entire time curves).



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Fig. 1. Effect of carboxymethylcellulose-linked phosphatidylethanolamine (CMPE) on intestinal permeation in rats with induced colon inflammation. Colon inflammation was induced by rectal administration of trinitrobenzenesulfonic acid (TNBS). The rats were treated with either vehicle (PBS) or CMPE, as described in MATERIALS AND METHODS. Inulin-fluorescein (InFl) was given rectally 12 h after TNBS, and its plasma level was determined 2 h later. The ordinate presents arbitrary units of fluorescence intensity (FU). Each datum is a mean ± SE for 6 rats. *P < 0.01.

 


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Fig. 2. Effect of CMPE on tissue injury of rats with TNBS-induced colitis. Rats with TNBS-induced colon injury were treated intraperitoneally with either vehicle (PBS) or CMPE, as described in MATERIALS AND METHODS, and were killed 48 h later. Intestinal sections were taken for histological examination by morphometry of tissue damage as described in MATERIALS AND METHODS. A: representative micrographs of colon section from a TNBS rat, a CMPE-treated rat, and a normal rat (for illustration of healthy intestine). B: corresponding histological morphometry for the TNBS and the CMPE-treated groups. Each datum in B is a mean ± SE for 5 rats.*P = 0.004.

 

To avoid interference of the assays with each other (e.g., InFl with PLA2 determination, cardiac puncture with MPO), each assay was performed with a separate set of rats. Due to our institution's regulations concerning use of experimental animals, the possibility of repeating experiments was limited, and the number of rats used in the above assays was determined primarily by the survival of the TNBS rats through the respective experiment (12 h for InFl permeation, 24 h for plasma PLA2, 48 h for MPO and survival).


    RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Effect of CMPE on mortality rate. Table 1, depicting the number of rats that survived the disease over the number of rats in each group, shows that treatment with the PLA2 inhibitor CMPE was markedly effective in reducing the mortality rate among the rats with TNBS-induced colitis.


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Table 1. Effect of CMPE on mortality rate in rats with induced colitis

 

Intestinal permeation. As noted in MATERIALS AND METHODS, the assessment of the effect of CMPE on the parameters of intestinal damage was obviously limited to the surviving animals, in which the related manifestations were less severe than in the rats that did not survive the experiment. However, in a separate study (25), we found that pathological intestinal permeation of InFl precedes the other markers of TNBS-induced colitis and reached its peak at ~12 h after induction of the disease (25). In TNBS-induced colitis, animals started dying as early as 24 h after induction of the disease. Therefore, to obtain data from all of the animals in the experiments, intestinal permeation was examined 12 h after administration of TNBS, when most of the untreated rats were still alive. This was done by rectal administration of InFl and determination of its plasma levels after 3 h as previously described (25). As shown in Fig. 1, treatment with CMPE maintained the intestinal permeation of InFl at a much lower level than in the untreated rats, suggesting that this treatment attenuates the development of the disease at an early stage.

Colon tissue damage. Naked-eye examination of colon of rats with TNBS-induced colitis revealed damage mainly in the mucosa, with no infiltration to the serosa, and this was practically ameliorated in the CMPE-treated rats. The corresponding macroscores (by the criteria described in MATERIALS AND METHODS) were 3.93 ± 0.43 and 1.19 ± 0.29 (n = 5; P < 0.001) for TNBS rats and CMPE-treated rats, respectively.

Figure 2A, depicting representative micrographs of colon tissue of rats with TNBS-induced colitis either left untreated or treated with CMPE and compared with normal rats, clearly demonstrates that the treatment with CMPE markedly reduced the TNBS-induced ulceration and maintained it practically at the level of normal rats. This is confirmed by the quantitative morphometry of histological damage, shown in Fig. 2B, depicting the colon ulcerative area for the untreated and CMPE-treated colitic rats.

Plasma PLA2 activity. To investigate the association of the disease with the level of plasma PLA2 activity, we monitored the time course of its activity in plasma of the CMPE-treated and untreated rats subsequent to induction of colitis by TNBS. Figure 3 shows that the level of plasma PLA2 activity is enhanced during the course of the disease and appears at an early stage after induction of the disease, considerably before the expression of intestinal permeation. Figure 3 further shows that treatment with the PLA2 inhibitor suppressed the elevation in plasma sPLA2 activity through the entire time course and brought it to the basal level within 24 h (and possibly earlier) after induction of disease.

MPO activity in intestinal tissue. MPO activity in inflamed tissues, which is a measure of inflammatory cell infiltration into the inflamed tissue (9) and is commonly taken as a measure of intestinal inflammation (14, 21), has been linked to PLA2 activity and shown to be suppressed by PLA2 inhibitors (14, 28). In accord with this, in the present study the treatment with CMPE considerably reduced the MPO activity in the colon of colitic rats. The respective MPO activity in the untreated and the CMPE-treated groups was 19.1 ± 2.6 and 7.9 ± 1.1 U/mg tissue (means ± SE; n = 5; P < 0.01).


    DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
The results of the present study demonstrate that treatment with the PLA2 inhibitor CMPE impressively attenuated the development of colon injury induced in rats. This was assessed by clinical (mortality rate, intestinal permeation), histological, and biochemical parameters (PLA2, MPO). As noted in the introduction, PLA2 activity has been reported to be involved in pathophysiology of intestinal inflammation. Previous studies have reported elevated expression of Group II sPLA2 (29) and its gene (18) in the mucosa of inflamed human intestine, and PLA2 isoenzyme is abundant in rats and other mammals as well. Inflammatory conditions are typically associated with elevated sPLA2 activity, secreted to body fluids by activated inflammatory cells and inflamed tissues. This is further supported in the present study, showing elevated plasma activity of PLA2 as determined by the hydrolysis of sPLA2-specific substrate. It is likely that the plasma PLA2 activity observed in the present study is secreted to body fluids from the injured colon, but since the same isoenzyme may be secreted from inflammatory cells on their activation, they can also contribute to the elevated plasma level of sPLA2 as measured in the present study. The ExPLIs have been shown to protect cells from activation and secretion of cellular sPLA2 induced by inflammatory stimuli, such as endotoxins (2, 4). Furthermore, they block subsequent cell-signaling processes, including NF-{kappa}B stimulation (4). Because this transcription factor is involved in the activation of the intracellular cPLA2, it is likely that the ExPLIs, although cell impermeable, affect a wider range of inflammatory processes beyond sPLA2.

As discussed in the introduction, PLA2 is responsible for the production of a host of inflammatory mediators and is involved in the development of inflammation directly and indirectly, mainly via eicosanoid production. In addition, inhibition of PLA2 and subsequent production of lysophospholipids suppress the production of PAF, which is a potent inflammatory agent and has been shown to be involved in IBD pathophysiology (40). The present study, showing that experimental injury of rat colon is ameliorated by ExPLI, strongly supports the therapeutic strategy of inclusive control of the production of inflammatory lipid mediators by inhibition of PLA2 activation.

Nonsteroidal anti-inflammatory drugs (NSAIDs), which are COX inhibitors, are commonly used in clinical practice but are often associated with gastrointestinal complications, such as ulceration, bleeding, and perforation, which may be life-threatening. Treatment with corticosteroid is also accompanied by numerous adverse effects. The type of PLA2 inhibitors used here introduces a novel approach to the treatment of IBD and presents a potential alternative prototype of NSAID for the treatment of IBD, with a protecting strategy against damage induced by the currently used NSAID and steroid treatments. As noted in the introduction, the ExPLIs have been found effective in amelioration of other inflammatory conditions, including bleomycin-induced lung injury in hamsters (4) and endotoxin-induced sepsis in rats (10). Together with the present study, these suggest that this kind of PLA2 inhibitor may be considered a plausible candidate for therapy of intestinal injury and other inflammatory diseases.


    DISCLOSURES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
This work was supported by grants to S. Yedgar and M. Krimsky from the Israel Ministry of Health (No. 4631) and the Hebrew University Intramural Research Fund.


    FOOTNOTES
 

Address for reprint requests and other correspondence: S. Yedgar Dept. of Biochemistry, Hebrew Univ.-Hadassah Medical School, Jerusalem, Israel 91120 (E-mail: yedgar{at}md2.huji.ac.il).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


    REFERENCES
 TOP
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 MATERIALS AND METHODS
 RESULTS
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 REFERENCES
 

  1. Amandi-Burgermeister E, Tibes U, and Kaiser BM, Friebe WG, and Scheuer WV. Suppression of cytokine synthesis, integrin expression and chronic inflammation by inhibitors of cytosolic phospholipase A2. Eur J Pharmacol 326: 237-250, 1997.[ISI][Medline]
  2. Balsinde J, Balboa MA, Yedgar S, and Dennis EA. Group V phospholipase A2 mediated oleic acid mobilization in lipopolysaccharide-stimulated P388D(1) macrophages. J Biol Chem 275: 4783-4786, 2000.[Abstract/Free Full Text]
  3. Banergee AK and Peters TJ. Experimental non-steroidal anti-inflammatory drug-induced enteropathy in the rat. Gut 31: 1358-1364, 1990.[Abstract]
  4. Beck GCh Hermes WC, Yard BA, Kaszkin M, Von Zabern D, Schulte J, Haak M, Prem K, Krimsky M, Van-Ackern K, Van-Der-Woude FJ, and Yedgar S. Amelioration of endotoxin-induced sepsis in rats by membrane-anchored lipid conjugates. Crit Care Med. In press.
  5. Beck GCh Yard BA, Schulte J, Oberacker R, Van-Ackern K, Van-Der-Woude FJ, Krimsky M, Kaszkin M, and Yedgar S. Inhibition of LPS-induced chemokine production in human lung endothelial cells by lipid conjugates anchored to the membrane. Br J Pharmacol 135: 1665-1674, 2002.[Abstract/Free Full Text]
  6. Benayahu D and Sela J. Histomorphometric analysis of heterotopic bone formed by stromal-osteogenic subpopulations. Calcif Tissue Int 59: 254-258, 1996.[ISI][Medline]
  7. Bertsch T and Aufenanger J. Phospholipase A2 in inflammatory bowel disease. Gut 41: 859-860, 1997.[Free Full Text]
  8. Blackwell GJ and Flower RJ. Inhibition of phospholipase. Br Med Bull 39: 260-264, 1983.[ISI][Medline]
  9. Bradley PP, Priebat DA, Christensen RD, and Rothstein G. Measurement of cutaneous inflammation: estimation of neutrophil content with an enzyme marker. J Invest Dermatol 78: 206-209, 1982.[Abstract]
  10. Breuer R, Lossos IS, Or R, Krimsky M, Dagan A, and Yedgar S. Abatement of bleomycin-induced pulmonary injury by cell-impermeable inhibitor of phospholipase A2. Life Sci 57: 237-240, 1995.
  11. Dan P, Dagan A, Krimsky M, Pruzanski W, Vadas P, and Yedgar S. Inhibition of type I and type II phospholipase A2 by cell-impermeable inhibitors. Biochemistry 37: 6199-6204, 1998.[ISI][Medline]
  12. Dan P, Nitzan DW, Dagan A, Ginsburg I, and Yedgar S. H2O2 renders cells accessible to lysis by exogenous phospholipase A2: a novel mechanism for cell damage in inflammatory processes. FEBS Lett 383: 75-78, 1996.[ISI][Medline]
  13. Eberhart CA and Dubois RN. Eicosanoids and the gastroenterological tract. Gastroenterology 109: 285-301, 1995.[ISI][Medline]
  14. Fabia R, Ar'Rajab A, Willen R, Andersson R, and Beng-mark S. Effect of putative phospholipase A2 inhibitors on acetic acid-induced acute colitis in the rat. Br J Surg 80: 1199-1204, 1993.[ISI][Medline]
  15. Ginsburg I and Kohen R. Cell damage in inflammatory and infectious sites might involve a coordinated "cross-talk" among oxidants, microbial hemolysins and amphiphiles, cationic proteins, phospholipases, fatty acids, proteinases and cytokines (an overview). Free Radic Res 22: 489-517, 1995.[ISI][Medline]
  16. Ginsburg I, Yedgar S, and Varani J. Diethyldithiocarbamate and nitric oxide synergize with oxidants and with membrane damaging agents to injure mammalian cells. Free Radic Res 27: 143-164, 1997.[ISI][Medline]
  17. Greaves MW and Camp RD. Prostaglandins, leukotrienes, phospholipase, platelet activating factor and cytokines: an integrated approach to inflammation of human skin. Arch Dermatol Res 280: 33-41, 1998.
  18. Haapamaki MM, Gronroos JM, Nurmi H, Alanen K, Kallajoki M, and Nevalainen TJ. Gene expression of group II phospholipase A2 in intestine in ulcerative colitis. Gut 40: 95-101, 1997.[Abstract]
  19. Hawkey CJ and Skelly MM. Gastrointestinal safety of selective COX-2 inhibitors. Curr Pharm Des 8: 1077-1089, 2002.[ISI][Medline]
  20. Heller A, Koch T, Schmeck J, and van-Ackern K. Lipid mediators in inflammatory disorders. Drugs 55: 487-496, 1998.[ISI][Medline]
  21. Hoshino H, Sygiyama S, Ohara A, Goto H, Tsukamoto Y, and Ozawa T. Mechanism and prevention of chronic inflammation with trinitrobenzene sulfonic acid in rats. Clin Exp Pharmacol Physiol 19: 717-722, 1992.[ISI][Medline]
  22. Ilan Y, Weksler-Zanger S, Ben-Horin S, Diment J, Sauter B, Rabbani E, Engelhardt D, Chowdhury NR, Chowdhury JR, and Goldin E. Treatment of experimental colitis by oral tolerance induction: a central role for suppressor lymphocytes. Am J Gastroenterol 95: 966-972, 2000.[ISI][Medline]
  23. Kates M. Techniques of Lipidology. New York: Elsevier, 1975, p. 347-353.
  24. Koike K, Moore EE, Moore FA, Carl VS, Pitman JM, and Banerjee A. Phospholipase A2 inhibition decouples lung injury from gut ischemia-reperfusion. Surgery 112: 173-180, 1992.[ISI][Medline]
  25. Krimsky M, Dagan A, Aptekar L, Ligumsky M, and Yedgar S. Determination of intestinal permeability in intestinal injury by permeation of inulin-fluorescein. J Basic Clin Physiol Pharmacol 11: 143-153, 2000.[Medline]
  26. Lauritsen K, Laursen LS, Bukhave K, and Rask-Madsen J. In vivo effects of orally administered prednisolone on prostaglandin and leukotriene production in ulcerative colitis. Gut 28: 1095-1099, 1987.[Abstract]
  27. Lauritsen K, Laursen LS, Kjeldsen K, Bukhave K, and Rask-Madsen J. Inhibition of eicosanoid synthesis and potential therapeutic benefits of "dual pathway inhibition." Pharmacol Toxicol 75: 9-13, 1994.[ISI][Medline]
  28. Marshall LA, Winkler JD, Griswold DE, Bolognese B, Roshak A, Sung CM, Webb EF, and Jacobs R. Effects of scala-radial, a type II phospholipase A2 inhibitor, on human neutrophil arachidonic acid mobilization and lipid mediator formation. J Pharmacol Exp Ther 268: 709-717, 1994.[Abstract]
  29. Minami T, Shinomura Y, Miyagawa J, Tojo H, Okamoto M, and Matsuzawa Y. Immunohistochemical localization of group II phospholipase A2 in colonic mucosa of patients with inflammatory bowel disease. Am J Gastroenterol 92: 289-292, 1997.[ISI][Medline]
  30. Peterson JW, Dickey WD, Saini SS, Gourley W, Klimpel GR, and Chopra AK. Phospholipase A2 activating protein and idiopathic inflammatory bowel disease. Gut 39: 698-704, 1996.[Abstract]
  31. Pfeilschiffer J, Schalwiek C, Briner VA, and Van den Bosch H. Cytokine-stimulated secretion of group II phospholipase A2 by rat mesangial cells. J Clin Invest 92: 2516-2523, 1993.[ISI][Medline]
  32. Pruzanski W, Stefanski E, Vadas P, Bukhave K, and Rask-Madsen J. Inhibition of extracellular release of pro-inflammatory secretory phospholipase A2 (sPLA2) by sulfasalazine: a novel mechanism of anti-inflammatory activity. Biochem Pharmacol 53: 1901-1907, 1997.[ISI][Medline]
  33. Pruzanski W and Vadas P. Phospholipase A2—a mediator between proximal and distal effectors of inflammation. Immunol Today 12: 143-146, 1991.[ISI][Medline]
  34. Rachmilewitz D, Eliakim R, Simon P, Ligumsky M, and Karmeli F. Cytokine and platelet-activating factor in human inflamed colonic mucosa. Agents Actions Spec No: C32-C36, 1992.
  35. Rachmilewitz D, Simon P, Schwartz LW, Griswold DE, Fondacaro JD, and Wasserman MA. Inflammatory mediators of experimental colitis in rats. Gastroenterology 97: 326-337, 1989.[ISI][Medline]
  36. Schnitzer E, Dagan A, Krimsky M, Lichtenberg D, Pinchuk I, Shinar H, and Yedgar S. Interaction of hyaluronic acid-linked phosphatidylethanolamine (HyPE) with LDL and its effect on the susceptibility of LDL lipids to oxidation. Chem Phys Lipids 104: 149-160, 2000.[ISI][Medline]
  37. Shakir KM. Phospholipase A2 activity of post-heparin plasma: a rapid and sensitive assay and partial characterization. Anal Biochem 114: 64-70, 1981.[ISI][Medline]
  38. Tanaka K and Arita H. Secretory phospholipase A2 inhibitors. Possible new anti-inflammatory agents. Agents Actions Suppl 46: 51-64, 1995.[Medline]
  39. Vadas P, Browning J, Edelson J, and Pruzanski W. Extracellular phospholipase A2 expression and inflammation: the relationship with associated disease states. J Lipid Mediators 8: 1-30, 1993.[ISI][Medline]
  40. Wallace JL and Ma L. Inflammatory mediators in gastrointestinal defense and injury. Exp Biol Med (Maywood) 226: 1003-1015, 2001.[Abstract/Free Full Text]
  41. Wardle TD, Hall L, and Turberg LA. Inter-relationships between inflammatory mediators released from colonic mucosa in ulcerative colitis and their effects on colonic secretion. Gut 34: 503-508, 1993.[Abstract]
  42. Wardle TD and Turnberg LA. Potential role for interleukin-1 in the pathophysiology of ulcerative colitis. Clin Sci (Colch) 86: 619-626, 1994.[ISI][Medline]
  43. Yang VW. Eicosanoids and inflammatory bowel disease. Gastroenterol Clin North Am 125: 317-332, 1996.
  44. Yard BA, Yedgar S, Scheele M, Van-der-Woude D, Beck G, Heidrich B, Krimsky M, Van-der-Woude FJ, and Post S. Modulation of IFN-gamma-induced immunogenicity by phosphatidylethanolamine-linked hyaluronic acid. Transplantation 73: 984-992, 2002.[ISI][Medline]
  45. Yedgar S, Dagan A, Dan P, Ginsburg I, Lossos IS, and Breuer R. Regulation of cell membrane phospholipase A2 activity by cell-impermeable inhibitors. In: Lipid Mediators in Health and Disease, edited by Zor U. London: Freund, 1994.
  46. Yedgar S and Gatt S. Effect of Triton X-100 on the hydrolysis of sphingomyelin by sphingomyelinase of rat brain. Biochemistry 15: 2570-2574, 1976.[ISI][Medline]
  47. Yedgar S, Lichtenberg D, and Schnitzer E. Inhibition of phospholipase A2 as a therapeutic target. Biochim Biophys Acta 1488: 182-187, 2000.[ISI][Medline]
  48. Yeomans N, Cook GA, and Giraud AS. Selective COX-2 inhibitors: are they safe for the stomach? Gastroenterology 115: 227-229, 1998.[ISI][Medline]