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Platelet-activating factor promotes mucosal apoptosis via FasL-mediating caspase-9 active pathway in rat small intestine after ischemia-reperfusion¹

Division of Gastroenterology, Department of Internal Medicine, Saga Medical School, Saga 849-8501, Japan

²Correspondence: Department of Internal Medicine and Gastrointestinal Endoscopy, Saga Medical School, 5-1-1 Nabeshima, Saga 849-8501, Japan. E-mail: fujimoto{at}post.saga-med.ac.jp

SPECIFIC AIMS

Growing evidence suggests that platelet-activating factor (PAF) plays an important role in initiation and amplification of ischemia-reperfusion (I/R) injury. PAF is a proinflammatory lipid mediator for inflammatory response; the initial step in the deactivation of PAF is its deacetylation by PAF-acetylhydrolase (PAF-AH). It is unclear whether and how PAF might involve mucosal apoptosis induced by I/R in the small intestine. The purpose of this study was to 1) examine whether the increase in PAF activity after I/R is caused by the inhibition of PAF-AH and 2) investigate whether PAF promotes mucosal apoptosis via the Fas-FasL-mediating caspase-9 active pathway in the small intestine after I/R.

PRINCIPAL FINDINGS

1. I/R-increased PAF activity through the inhibition of PAF-AH in small intestinal mucosa
PAF was extracted and biological activity was analyzed by a platelet aggregation system. PAF-AH activity was determined by a PAF-AH assay. After small intestines were subjected to 60 min of ischemia with occlusion of the superior mesenteric artery followed by a 60 min of reperfusion, jejunal and ileal mucosal PAF activities increased significantly compared with the sham-operated rats (Fig. 1 A). Jejunal and ileal mucosal PAF-AH activities decreased significantly compared with sham-operated rats (Fig. 1B ). The results suggest that I/R increased the PAF activity through inhibition of PAF-AH.

View larger version (15K): [in this window] [in a new window]   Figure 1. I/R increased the mucosal PAF activity (A). In contrast, I/R inhibited the mucosal PAF-AH activity (B). a: P < 0.01 compared with sham-operated rats.

2. PAF promoted I/R-induced mucosal apoptosis via Fas ligand (FasL)-mediating pathway in the small intestine
To investigate the effects of PAF on mucosal apoptosis induced by I/R in the small intestine, apoptosis was evaluated by DNA fragmentation assay and terminal deoxynucleotidyl transferase (TdT)-mediated dUDP-biotin nick end label (TUNEL) staining. The percentage of mucosal fragmented DNA to total DNA increased significantly in the I/R rats pretreated with the vehicle (jejunum: 32.3±1.4, ileum: 31.1±1.9) compared with sham-operated rats (jejunum: 4.5±0.4, ileum: 4.0±0.3). The increase was reduced by pretreatment with the PAF antagonist 40 mg/kg BN-52021 (jejunum: 20.9±2.1, ileum: 19.6±1.8). The effect of BN-52021 on the apoptosis was dose dependent (20–40 mg/kg), but a continuously increasing dose of BN-52021 (80 mg/kg) had no further attenuation effect on apoptosis than a dose of 40 mg/kg. The I/R-induced mucosal apoptosis was significantly suppressed by pretreated with 0.2 mg/kg anti-FasL antibody (jejunum: 15.1±2.1, ileum: 12.2±1.5). TUNEL staining of the small intestine showed an increase in the number of apoptotic cells and marked destruction of the structure with mucosal erosion and edema in the I/R rats. Pretreatment of BN-52021 (40 mg/kg) significantly decreased the number of apoptotic cells and the destruction of the structure of the intestinal mucosa in the I/R rats. The increased apoptotic index after I/R was attenuated by pretreatment of the PAF antagonist.

3. PAF enhanced inflammatory interleukin-6 (IL-6) secretion and inhibited anti-inflammatory IL-10 synthesis in the small intestinal mucosa after I/R
Mucosal IL-6 and IL-10 concentrations were evaluated by ELISA. The jejunal and ileal mucosal IL-6 concentrations increased significantly after I/R, and this increase was suppressed by pretreatment with BN-52021 (40 mg/kg). The decrease in the mucosal IL-10 concentration after I/R returned to control level by pretreatment with BN-52021 (40 mg/kg).

4. PAF enhanced the expression of platelet endothelial cell adhesion of molecule-1 (PECAM-1) and FasL, but not Fas, and activated caspase-9 in the small intestinal mucosa after I/R
In sham-operated rats, small amounts of PECAM-1, Fas, and FasL were detected by Western blot in membranous fraction. Amounts of PECAM-1, Fas, and FasL in small intestinal mucosa increased significantly after I/R, and the increases in PECAM-1 and FasL (but not Fas) were suppressed by pretreatment with the PAF antagonist (Fig. 2 A–C). Bid, cytochrome c, and caspase-9 were detected in the cytosolic fraction. Cleavage of Bid, the release of cytochrome c from mitochondria, and the expression of cleaved activated caspase-9 were significantly increased in the intestinal mucosa after I/R, and the increase was inhibited by pretreatment with the PAF antagonist (Fig. 2D-F ). The results indicate that PAF enhanced the expression of PECAM-1 and FasL, but not Fas, and induced the cleavage of Bid, resulting in the release of cytochrome c from mitochondria to activate caspase-9.

View larger version (40K): [in this window] [in a new window]   Figure 2. PAF enhanced the expression of PECAM-1 and FasL, but not Fas, induced cleavage of Bid, the release of cytochrome c from mitochondria, and activation of caspase-9 in small intestinal mucosa after I/R. A) PECAM-1; B) FasL; C) Fas; D) Bid and tBid; E) cytosolic cytochrome c; F) procaspase-9 and cleaved caspase-9. Results are expressed as a ratio to ß-actin densitometry units. Lanes 1–3, sham-operated rats, I/R rats pretreated with vehicle, I/R rats pretreated with BN-52021 (40 mg/kg), respectively. a: P < 0.01 compared with sham-operated rats. b: P < 0.01 compared with I/R rats pretreated with the vehicle.

CONCLUSIONS AND SIGNIFICANCE

I/R increased PAF activity through the inhibition of PAF-AH in rat small intestinal mucosa; the I/R increased mucosal inflammatory modulator IL-6 concentration and reduced anti-inflammatory cytokine IL-10 synthesis. Pretreated with the PAF antagonist, this increase in IL-6 was suppressed, and the concentration of IL-10 was returned to the control level. Pretreatment of the PAF antagonist suppressed I/R-induced small intestinal mucosal apoptosis. The results indicate that the I/R-inhibited mucosal PAF-AH activity resulted in the loss of PAF inactivation and that activated PAF was involved in the I/R induced mucosal apoptosis in the small intestine by enhancing IL-6 secretion and inhibiting IL-10 synthesis.

Several indications suggest that PECAM-1 is necessary for leukocyte transmigration through endothelial cell monolayers and acts as a signaling molecule in apoptotic induction or inhibition. The stimuli that initiate the apoptotic process include extracellular signals such as the Fas and FasL. Stimulation of the Fas pathway in response to FasL results in recruitment of proteins that form the death-inducing signaling complex, including procaspase-8 and the Fas-associated death domain. There are two intracellular signal pathways in Fas-mediated apoptosis, including type I cell death and type II cell death. In type I cell death, local aggregation of procaspase-8 is sufficient to produce active caspase-8, which can subsequently activate executioners. In type II cell death, small amounts of active caspase-8 cleave Bid, resulting in the release of cytochrome c from mitochondria. In the present study, we demonstrated that pretreatment with anti-FasL antibody significantly suppressed I/R-induced mucosal apoptosis in small intestine. We found that I/R induced expression of PECAM-1, Fas, and FasL proteins, cleavage of Bid, release of cytochrome c from mitochondria, and the expression of cleaved activated caspase-9 in small intestinal mucosa. Pretreatment of PAF antagonist inhibited this expression of PECAM-1 and FasL, but not Fas, and suppressed cleavage of Bid, release of cytochrome c, and activation of caspase-9. The results suggest that activated PAF enhanced the expression of PECAM-1 and secretion of the inflammatory cytokine IL-6 and inhibited the synthesis of anti-inflammatory cytokine IL-10. These changes increased FasL, but not Fas. FasL binding to Fas led to cleavage of Bid, resulting in the release of cytochrome c from mitochondria to cytosols in order to activate caspase-9 (Fig. 3 ). The PAF promoted mucosal apoptosis, at least in part, via the FasL-mediating caspase-9 active pathway in rat small intestine after I/R. Our previous study showed that I/R-induced small intestinal mucosal apoptosis did not occur through the caspase-3 pathway. It is unclear whether caspase-9 induced apoptosis through activation of caspase-6 or/and -7 or through a direct effect on the poly-ADP-ribose polymerase.

View larger version (56K): [in this window] [in a new window]   Figure 3. Schematic diagram of PAF promotes I/R-induced mucosal apoptosis in small intestine. I/R inhibit PAF-AH activity results in an increase in activated PAF. Activated PAF enhances expression of PECAM-1 and secretion of IL-6 and inhibits the synthesis of IL-10, inducing an increase in FasL but not in Fas. FasL binding Fas led to a cleavage of Bid, resulting in a release of cytochrome c from mitochondria to cytosols to activate caspase-9; the activated caspase-9 increases mucosal apoptosis.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0499fje; to cite this article, use FASEB J. (April 22, 2003) 10.1096/fj.02-0499fje

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