Shigeaki Morooka

The role of P-selectin, sialyl Lewis X and sulfatide in myocardial ischemia and reperfusion injury

The role of P-selectin and the ligands of selectins such as sialyl Lewis X and sulfatide was studied in a myocardial ischemia and reperfusion injury model. Anesthetized rabbits underwent the occlusion of coronary artery (30 min) followed by reperfusion (5 h). The inhibitory effect on myocardial ischemia and reperfusion injury was examined with infarct size normalized by area-at-risk. Intravenous administration of an anti-P-selectin monoclonal antibody, PB1.3 (2 mg/kg), reduced infarct size by 38%. Similarly, the administration of sialyl Lewis X-oligosaccharide (10 mg/kg) reduced infarct size by 53% significantly. Finally, the infarct size was significantly reduced bv 39% in sulfatide-treated group (10 mg/kg). These results suggest that P-selectin plays an important role in myocardial ischemia and reperfusion injury and that the ligands of selectins, such as sialyl Lewis X-oligosaccharide and sulfatide, have cardioprotective effect on myocardial ischemia and reperfusion injury.

Role of P-selectin in the early stage of the Arthus reaction

P-selectin is rapidly translocated to the surface of endothelial cells and platelets following exposure to chemical mediators such as histamine, thrombin and complement factors. The Arthus reaction is caused by vascular injury which is initiated by the local deposition of the immune complex followed by the activation of complement and release of chemical mediators. In this report, the role of P-selectin in the early stage of reverse passive Arthus reaction in rat using monoclonal antibodies (mAbs) against rat P-selectin will be investigated. Intravenous administration of the mAb ARP2-4 significantly attenuated paw edema 1 h after challenging it with antigen by 31.5% (1 mg/kg) and 44.7% (3 mg/kg), respectively. Edema formation was also reduced by sulfatide (73.1%, 50 mg/kg) and inositol hexakisphosphate (InsP6) (72.9%, 30 mg/kg), which have been reported to block P-selectin-mediated neutrophil adhesion. Moreover, neutrophil accumulation into the inflammatory site in the Arthus reaction was inhibited by anti-P-selectin mAb. P-selectin expression was detected along vessel walls prior to neutrophil accumulation, as determined by immunohistochemical staining using the antibody. In addition, the expression of P-selectin mRNA was induced 4 h after deposition of immune complex. From these results, we concluded that P-selectin plays an important role in the pathogenesis of the Arthus reaction especially in the early stage by recruiting neutrophils into sites of inflammation.

Platelet-activating factor (PAF) plays an important role in the immediate asthmatic response in guinea-pig by augmenting the response to histamine.

1 To investigate the role of platelet activating factor (PAF) in the immediate asthmatic response, we examined the bronchial reactivity to histamine after administration of PAF to guinea‐pigs or antigen challenge to passively sensitized guinea‐pigs. 2 A bolus injection of PAF (20–40 ng kg−1), which did not cause a significant increase in intrathoracic pressure (ITP), augmented the bronchial response to histamine almost 8 fold. This airway hyperreactivity was observed even 1 min after PAF treatment. 3 A subthreshold dose of antigen (0.01 mg kg−1, i.v.) also provoked hyperreactivity to histamine, which became significant 6 and 11 min after the antigen treatment. 4 The specific PAF‐antagonists, SM‐10661 and CV‐6209 (i.v.) dose‐dependently inhibited both PAF‐and antigen‐induced airway hyperreactivities to histamine. 5 These results suggest that PAF plays an important role in antigen‐induced acute airway responses by augmenting the activities of spasmogens.

Effect of a selective PAF antagonist SM-10661 ((+/-)-cis-3,5-dimethyl-2-(3-pyridyl)thiazolidin-4-one HCl) on experimental disseminated intravascular coagulation (DIC).

Intravenous infusion of endotoxin (0.25 mg/kg/hr for 4 hr) was shown to induce disseminated intravascular coagulation (DIC) in rats, which resulted in hypofibrinogenemia, prolongation of prothrombin (PT) and partial thromboplastin time (PTT), thrombocytopenia, and elevated levels of fibrinogen/fibrin degradation products (FDP). Oral administration (100 mg/kg) of the selective PAF antagonist, SM-10661 ((±)-cis-3,5-dimethyl-2-(3-pyridyl)thiazolidin-4-one HCl), counteracted the changes caused by the endotoxin. Intravenous infusion of SM-10661 (6mg/kg bolus 2 min before endotoxin infusion+6 mg/kg/hr for 4 hr infusion) also counteracted DIC. When suboptimal doses of gabexate mesilate, a synthetic protease inhibitor (3 mg/kg i.p.), and SM-10661 (2 mg/kg bolus + 2 mg/kg/hr for 4 hr infusion) were administered concomitantly, hematological parameters improved. The results suggest that PAF may play a role in the pathogenesis of DIC, and that together with the results already reported for other PAF antagonists, SM-10661 may be useful in the treatment of DIC.

Inhibition of P-selectin attenuates neutrophil-mediated myocardial dysfunction in isolated rat heart

The expression of P-selectin on postischemic endothelium after reperfusion has been shown to trigger neutrophil attachment and the subsequent inflammatory responses. Extensive studies have demonstrated that P-selectin is involved in the progression of neutrophil-mediated myocardial infarction and no-reflow phenomenon. In the present study, we examined the effects of selectin inhibitors, sialyl Lewis X-oligosaccharide and anti-P-selectin monoclonal antibody, PB1.3 on neutrophil-dependent left ventricular dysfunction in isolated rat heart. The hearts were subjected to global ischemia for 20 min and then reperfused for 45 min with rat plasma in the presence of human neutrophils during the first 5 min of the reperfusion. Left ventricular developed pressure and other parameters of the left ventricular function deteriorated throughout the reperfusion period in a neutrophil-dependent manner. In contrast, the coronary flow was reduced early on (< 15 min) but recovered to the level in the hearts reperfused with no neutrophils 45 min after the reperfusion. We examined the effects of selectin inhibitors under experimental conditions in which the hearts were perfused with 30 million neutrophils. The treatment with sialyl Lewis X-oligosaccharide at a dose of 0.3 mg/min resulted in amelioration of left ventricular developed pressure to 57.2 +/- 14%, compared to 26.1 +/- 4.3% in the saline-treated group (P < 0.05). Similarly, the treatment with mouse anti-human P-selectin monoclonal antibody (IgG1) PB1.3 at a dose of 0.6 mg/min resulted in the prominent recovery of left ventricular developed pressure after 45 min of reperfusion (59.9 +/- 9.3% vs. 26.1 +/- 4.3% in the saline-treated group, P < 0.05). PB1.3 also attenuated the elevation of left ventricular end-diastolic pressure compared to that of the saline-treated group during the reperfusion period. Moreover, the treatment with PB1.3 ameliorated the recovery of coronary flow until 10 min after the reperfusion and the recovery of coronary flow 10 min after the reperfusion was 55.2 +/- 9.2%, as compared to 28.2 +/- 7.7% in saline-treated hearts (P < 0.05). To our knowledge, this is the first direct demonstration that the specific inhibition of P-selectin results in the inhibition of neutrophil-mediated left ventricular dysfunction or myocardial stunning.

Effect of the selective PAF antagonist SM-10661 on an asthmatic model. 2. Effect on antigen-induced dual asthmatic response and infiltration of leukocytes into airways in actively sensitized conscious guinea pigs

The effect of a selective platelet-activating factor (PAF) receptor antagonist, SM-10661, on antigen-induced dual asthmatic response and leukocyte infiltration into the airways of actively sensitized conscious guinea pigs was investigated. The animals were pretreated with mepyramine maleate (10 mg/kg i.p.) and then challenged with ovalbumin. The inhalation of ovalbumin caused an immediate decline in specific airway conductance (sGaw) which peaked at 5 min after the challenge. sGaw gradually returned to the baseline 6 hr after the challenge. After the early asthmatic response (EAR), a second phase change, a late asthmatic response (LAR) in sGaw peaking at 17–20 hr was observed. When 1% w/v disodium cromoglycate was inhaled for 2 min on two occasions, EAR was not affected, but LAR was significantly inhibited. Oral administration of 50 mg/kg fenoterol (30 min before challenge) significantly inhibited EAR, but had no significant effect on LAR. SM-10661 administered orally 1 hr before the challenge inhibited EAR dose-dependently (50% inhibitory dose (ID50); 59 mg/kg, but was even more effective against the LAR (ID50); 13–16 mg/kg). When 30 mg/kg of SM-10661 was administered orally 6 hr after ovalbumin challenge, LAR was completely inhibited. The number of total cells, macrophages and eosinophils in bronchoalveolar lavage fluids rose significantly 17 hr after antigen challenge compared to that observed after saline challenge. The number of neutrophils and lymphocytes also increased in response to the ovalbumin challenge, but not significantly. SM-10661 (30 mg/kg) administered orally 1 hr before the challenge significantly inhibited the increase in total cells, macrophages and eosinophils. By comparison, when 30 mg/kg SM-10661 was administered orally 6 hr after the challenge, these increases were not significantly inhibited. These results suggest that PAF is involved in EAR and LAR, and that SM-10661 may be a useful therapeutic agent for the treatment of allergic asthma.

Effect of the Selective PAF Antagonist SM-10661 on an Asthmatic Model. 1. Effect on Passive Anaphylactic Bronchoconstriction in Guinea Pigs'

The effect of SM-10661, a selective antagonist of platelet-activating factor (PAF), on passive anaphylactic bronchoconstriction was examined in guinea pigs. A challenge of ovalbumin to passively sensitized guinea pigs induced bronchoconstriction, which peaked at 4 min. When SM-10661 was administered intravenously 2 min before ovalbumin challenge, bronchoconstriction was inhibited dose-dependently with an ID50 of 68 mg/kg. In guinea pigs pretreated with 15 μg/kg mepyramine which is a suboptimal dose, antigen-induced bronchoconstriction peaked at 4–6 min, but was inhibited by SM-10661 with an ID50 of 21 mg/kg. When guinea pigs were pretreated intravenously with 2.5 mg/kg mepyramine, 1 mg/kg indomethacin and 0.01 mg/kg propranolol, the antigen-induced bronchoconstriction peaked at 6 min. SM-10661 inhibited the response with an ID50 of 45 mg/kg. Histamine- and leukotriene D4-induced bronchoconstrictions were unaffected by up to 100 mg/kg SM-10661. Ovalbumin challenge of minced lungs from passively sensitized guinea pigs triggered the release of leukotrienes and histamine. SM-10661 had no effect on the antigen-induced release of peptide leukotrienes or histamine up to 10−4 M. These results indicate that SM-10661 may be a useful tool to investigate the role of PAF in antigen-induced anaphylactic bronchoconstriction.

Effect of SM-12502 on Disseminated Intravascular Coagulation (DIC) in Tumor-Bearing Rats

To investigate the role of PAF in tumor-associated disseminated intravascular coagulation (DIC), we have established the tumor-induced DIC model in rats and examined the effect of PAF-antagonist as compared with commonly used anti-DIC drugs. Four days after the intraperitoneal inoculation of rat ascites hepatoma AH-130, DIC-like hematological parameter changes such as decrease in platelet count, prolongation of PTT and increase in FDP were observed. In addition, serum levels of GOT and GPT were increased, indicating that liver injury was provoked. PAF-antagonist SM-12502 inhibited the development of DIC (PT, PTT and FDP) and liver injury. These results indicate that PAF plays an important role in tumor-associated DIC as well as accompanying organ failure.