Essential role of P-selectin in the initiation of the inflammatory response induced by hemorrhage and reinfusion.

Resuscitation from hemorrhage induces profound pathophysiologic alterations and activates inflammatory cascades able to initiate neutrophil accumulation in a variety of tissues. This process is accompanied by acute organ damage (e.g., lungs and liver). We have previously demonstrated that significant leukocyte-endothelium interactions occur very early in other forms of ischemia/reperfusion (i.e., splanchnic ischemia/reperfusion and traumatic shock) which are largely mediated by increased expression of the adhesion molecule, P-selectin, on the vascular endothelium. Here we postulated that increased endothelial expression of P-selectin in the microvasculature would play an essential role in initiating the inflammatory signaling of hemorrhagic shock. Using intravital microscopy, we found that hemorrhagic shock significantly increased the number of rolling and adherent leukocytes in the mouse splanchnic microcirculation. In contrast, mice genetically deficient in P-selectin, or wild-type mice given either an anti-P-selectin monoclonal antibody or a recombinant soluble P-selectin glycoprotein ligand (PSGL)-1 immunoglobulin, exhibited markedly attenuated leukocyte-endothelium interaction after hemorrhagic shock. Thus, activation of P-selectin protein on the microvascular endothelium is essential for the initial upregulation of the inflammatory response occurring in hemorrhagic shock. Moreover, endogenous levels of PSGL-1 mRNA were significantly increased in the lung, liver, and small intestine of wild-type mice subjected to hemorrhagic shock. Since PSGL-1 promotes adhesive interactions largely through P-selectin expressed on the vascular endothelium, this result further supports the crucial role played by P-selectin in the recruitment of leukocytes during hemorrhagic shock.

Mediation of cardioprotection by transforming growth factor-beta.

Myocardial ischemia causes heart injury that is characterized by an increase in circulating tumor necrosis factor (TNF), the local production of superoxide anions, the loss of coronary vasodilation (relaxation) in response to agents that release endothelial cell relaxation factor, and cardiac tissue damage. Ischemic injury can be mimicked by TNF. When given before or immediately after ischemic injury, transforming growth factor-beta (TGF-beta) reduced the amount of superoxide anions in the coronary circulation, maintained endothelial-dependent coronary relaxation, and reduced injury mediated by exogenous TNF. Thus, TGF-beta prevented severe cardiac injury, perhaps by alleviating damage mediated by increases in circulating TNF.

Role of platelet-activating factor-acether in mediating guinea pig anaphylaxis.

The pathophysiology of anaphylaxis is very complex, and the sequelae of events are not fully explained in terms of the effects of histamine and peptide leukotrienes alone. Platelet-activating factor (1-O-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine, PAF-acether) has been detected in animals undergoing anaphylaxis. Injection of synthetic PAF-acether induces similar effects, including bronchoconstriction, respiratory arrest, systemic hypotension, neutropenia, and thrombocytopenia. The results reported here demonstrate that the histamine- and leukotriene-independent component of guinea pig anaphylaxis in vivo and in isolated lung parenchymal strips in vitro is mediated by PAF-acether. However, PAF-acether is not responsible for the anaphylaxis-induced thrombocytopenia.

Prostacyclin: a potentially valuable agent for preserving myocardial tissue in acute myocardial ischemia.

Prostacyclin, a potent, naturally occurring prostaglandin exerts a variety of cardiovascular and cellular actions of potential value in acute myocardial ischemia. These properties include the reduction of systemic blood pressure without changing heart rate, the lowering of coronary vascular and total peripheral resistance, the inhibition of platelet aggregation and the concomitant formation of thromboxane B2, and the reduction of the release of lysosomal enzymes.

Antibody to CD-18 exerts endothelial and cardiac protective effects in myocardial ischemia and reperfusion.

We studied the effects of MAbR15.7, an antibody directed against the common beta-chain (CD-18) of a family of neutrophil adherence glycoproteins, on endothelial dysfunction and myocardial injury in a model of myocardial ischemia and reperfusion in cats. Pentobarbital-anesthetized cats were subjected to 1.5 h occlusion of the left anterior descending coronary artery (LAD) and 4.5 h of reperfusion. MI + R resulted in severe myocardial injury and endothelial dysfunction, including significant elevation of plasma creatine kinase (CK) activity, marked myocardial necrosis, high cardiac myeloperoxidase (MPO) activity in ischemic cardiac tissue, and loss of response of LAD coronary rings to the endothelium-dependent vasodilators, acetylcholine (ACh) and A-23187. In contrast, MAbR15.7-treated cats exhibited a lower plasma CK activity at every time point observed after 2 h, a reduced area of cardiac necrosis (2 +/- 1 vs. 30.8 +/- 2.5% of area-at-risk, P less than 0.001), lower MPO activity in the ischemic region (P less than 0.01), and significantly preserved vasorelaxant responses of LAD coronary rings to endothelium-dependent vasodilators, ACh (P less than 0.001), and A-23187 (P less than 0.001). These results indicate that myocardial ischemia and reperfusion induces significant myocardial injury and endothelial dysfunction in the cat involving a CD18-dependent neutrophil adherence mechanism. Inhibition of neutrophil adherence to the endothelium exerts significant protective effects in this model of reperfusion injury.

Cardiovascular effects of acute hypercholesterolemia in rabbits. Reversal with lovastatin treatment.

Hypercholesterolemia was induced in New Zealand white rabbits by feeding them a 0.5% cholesterol-enriched rabbit chow for 2 wk. Half of the cholesterol-fed rabbits were given lovastatin, a potent inhibitor of hydroxymethylglutaryl-coenzyme A reductase (HMG-CoA reductase), the rate limiting enzyme in cholesterol biosynthesis, and the other half were given its vehicle (i.e., DMSO). At the end of 2 wk, the rabbits underwent experimental myocardial ischemia or a sham ischemia procedure. Ischemic animals fed the cholesterol-enriched diet for 2 wk experienced much greater cardiac damage than ischemic rabbits fed the control diet, despite the absence of any atherosclerosis. Lovastatin was shown to protect the ischemic rabbit myocardium by three different indices of ischemic damage: (a) maintenance of creatine kinase (CK) activity in the ischemic myocardium; (b) reduced loss of free amino-nitrogen containing compounds from the ischemic myocardium; and (c) blunting the rise of plasma CK activity. These effects were not due to differences in myocardial oxygen demand between the groups. Arteries isolated from animals fed the cholesterol-enriched diet developed defects in endothelium-dependent relaxation in both large vessels as well as coronary resistance vessels. Acute hypercholesterolemia increases the severity of myocardial ischemia while at the same time impairing endothelium-dependent relaxation. These deleterious changes can be significantly attenuated by treatment with lovastatin.

In vivo neutralization of P-selectin protects feline heart and endothelium in myocardial ischemia and reperfusion injury.

The cardioprotective effects of an mAb to P-selectin designated mAb PB1.3 was examined in a feline model of myocardial ischemia (MI) and reperfusion. PB1.3 (1 mg/kg), administered after 80 min of ischemia (i.e., 10 min before reperfusion), significantly attenuated myocardial necrosis compared to a non-blocking mAb (NBP1.6) for P-selectin (15 +/- 3 vs 35 +/- 3% of area at risk, P < 0.01). Moreover, endothelial release of endothelium derived relaxing factor, as assessed by relaxation to acetylcholine, was also significantly preserved in ischemic-reperfused coronary arteries isolated from cats treated with mAb PB1.3 compared to mAb NBP1.6 (67 +/- 6 vs 11 +/- 3, P < 0.01). This endothelial preservation was directly related to reduced endothelial adherence of PMNs in ischemic-reperfused coronary arteries. Immunohistochemical localization of P-selectin was significantly upregulated in the cytoplasm of endothelial cells that lined coronary arteries and veins after 90 min of ischemia and 20 min of reperfusion. The principal site of intracytoplasmic expression was in venous vessels. mAb PB1.3 significantly decreased (P < 0.01) adherence of unstimulated PMNs to thrombin and histamine stimulated endothelial cells in a concentration-dependent manner in vitro. These results demonstrate that PMN adherence to endothelium by P-selectin is an important early consequence of reperfusion injury, and a specific monoclonal antibody to P-selectin exerts significant endothelial preservation and cardioprotection in myocardial ischemia and reperfusion.

Peroxynitrite inhibits leukocyte-endothelial cell interactions and protects against ischemia-reperfusion injury in rats.

Peroxynitrite (ONOO-) anion, formed by the interaction of superoxide with nitric oxide (NO), has previously been implicated as a cytotoxic agent. However, the effects of this free radical species on neutrophil (PMN)-endothelial cell interactions is largely unknown. We investigated the direct actions of ONOO- on PMN adhesion to endothelial cells in vitro and in vivo, as well as the effects of ONOO- on PMN-mediated myocardial ischemia-reperfusion injury. In vitro, peroxynitrite (100-1,000 nM) inhibited the adhesion of rat PMNs to the endothelium of isolated thrombin- or H2O2-stimulated rat mesenteric artery (P < 0.01 vs. thrombin or H2O2 alone). In vivo, in the rat mesentery, thrombin (0.5 U/ml) or N(G)-nitro-L-arginine-methyl ester (50 microM) significantly increased venular leukocyte rolling and adherence, which were also significantly (P < 0.01) attenuated by ONOO (800 nM) accompanied by reduced P-selectin expression on the endothelial cell surface. Isolated perfused rat hearts were subjected to global ischemia and reperfusion with rat PMNs (10(8) cells), which resulted in profound cardiac depression (i.e., a marked reduction in left ventricular developed pressure and maximal rate of development of left ventricular pressure). Infusion of ONOO- reversed the myocardial contractile dysfunction of ischemic-reperfused rat hearts to near baseline levels, and markedly attenuated the accumulation of PMNs in the postischemic heart. The present study provides strong evidence that nanomolar concentrations of ONOO- both inhibit leukocyte-endothelial cell interactions and exert cytoprotective effects in myocardial ischemia-reperfusion injury. Furthermore, our results suggest that the inhibition of P-selectin expression by peroxynitrite is a key mechanism of the modulatory actions of ONOO- on leukocyte-endothelial cell interactions.

Sialyl Lewisx-containing oligosaccharide attenuates myocardial reperfusion injury in cats.

Neutrophil (PMN) adhesion to the vascular endothelium is an important mechanism of myocardial reperfusion injury. The adhesion process is initially mediated by selectins (e.g., P- and L-selectin), and monoclonal antibodies directed against these adhesion molecules exert cardioprotective activity in ischemia/reperfusion models. The counterreceptors for these selectins are thought to be carbohydrate-containing moieties. In this connection, we studied the effect of a soluble sialyl Lewisx-containing oligosaccharide (SLex-OS) on PMN-endothelial interactions in a feline model of myocardial ischemia/reperfusion (MI/R). SLex-OS (10 mg/kg), administered 10 min before R, significantly reduced myocardial necrosis compared with its vehicle 270 min after reperfusion (6 +/- 1% vs. 35 +/- 4% of area at risk, P < 0.01). The cardioprotection was confirmed by significantly lower plasma creatine kinase activities in SLex-OS vs. vehicle-treated cats (P < 0.01). Cardiac contractility (dP/dt max) of cats receiving SLex-OS was significantly preserved after 270 min of R (97 +/- 2% vs. 78 +/- 5% of initial, P < 0.01). Furthermore, endothelium-dependent relaxation to acetylcholine in coronary artery rings isolated from MI/R cats treated with SLex-OS was significantly preserved (73 +/- 7% vs. 22 +/- 6% vasorelaxation, P < 0.01). In vitro PMN adherence to coronary vascular endothelium after 270 min of R was significantly attenuated in the SLex-OS-treated group compared with the vehicle group (14 +/- 5 vs. 91 +/- 12 PMN/mm2, P < 0.01). Our results indicate that a SLex-OS is cardioprotective and preserves coronary endothelial function after MI/R, indicating an important role of sialyl Lewisx in PMN accumulation, endothelial dysfunction, and myocardial injury in myocardial ischemia/reperfusion.

Isolation of myocardial depressant factor from plasma of dogs in hemorrhagic shock.

Starting with 31 of plasma from dogs in hemorrhagic shock, we have purified the myocardial depressant factor and found that the activity is separate from salts and free amino acids. Moreover, the myocardial depressant factor is present in shock plasma in concentrations of about 1 nmol/ml of plasma. The depressant factor exists as multiple chromatographic forms. The best characterized forms are the anionic forms. A preliminary amino acid composition of the anionic forms has been obtained. These findings should allow more rapid processing of plasma containing high myocardial depressant factor activity to separate the factor and to completely identify this small peptide of great physiologic interest.

Simvastatin preserves the ischemic-reperfused myocardium in normocholesterolemic rat hearts.

BACKGROUND: Ischemia followed by reperfusion in the presence of polymorphonuclear leukocytes (PMNs) results in cardiac contractile dysfunction as well as cardiomyocyte injury. These deleterious effects are due in large part to endothelial dysfunction leading to the upregulation of cell adhesion molecules and subsequent neutrophil-endothelium interaction. At clinically relevant doses, simvastatin, an HMG-CoA reductase inhibitor, has been shown to lower serum cholesterol levels and normalize endothelial cell function. We wanted to test the effects of simvastatin on neutrophil-mediated cardiac dysfunction in a controlled model of myocardial ischemia-reperfusion. METHODS AND RESULTS: This study examines the effects of simvastatin in a neutrophil-dependent isolated perfused rat heart model of ischemia (I) (20 minutes) and reperfusion (R) (45 minutes) injury. Administration of simvastatin 25 micrograms/rat improved coronary flow and preserved left ventricular developed pressure (LVDP) and dP/dtmax, indexes of cardiac contractile function. Final LVDP was 95+/-5 mm Hg in I/R hearts perfused with PMNs and simvastatin, compared with 49+/-4 mm Hg in PMN-perfused I/R hearts receiving only vehicle (P<0.001). In addition, simvastatin significantly reduced PMN accumulation in the ischemic myocardium (P<0.01). In PMN-perfused rat hearts after I/R, simvastatin also significantly attenuated P-selectin expression, CD18 upregulation in rat PMNs, and PMN adherence to rat vascular endothelium. Significant, although less potent, effects were obtained with pravastatin. CONCLUSIONS: These results provide evidence that HMG-CoA reductase inhibitors are potent and effective cardioprotective agents that inhibit leukocyte-endothelial cell interactions and preserve cardiac contractile function and coronary perfusion after myocardial ischemia and reperfusion. Moreover, these effects are unrelated to the cholesterol-lowering action of this agent and appear to be mediated by enhanced endothelial release of NO.

Simvastatin exerts both anti-inflammatory and cardioprotective effects in apolipoprotein E-deficient mice.

BACKGROUND: Simvastatin attenuates ischemia and reperfusion in normocholesterolemic animals by stabilizing endothelial nitric oxide synthase activity and inhibiting neutrophil-mediated injury. Because endothelial dysfunction is a detrimental effect of hypercholesterolemia, we examined whether short-term treatment with simvastatin could inhibit leukocyte-endothelium interaction and attenuate myocardial ischemia-reperfusion injury in apoE-deficient (apoE(-/-)) mice fed a high-cholesterol diet. METHODS AND RESULTS: We studied leukocyte-endothelium interactions in apoE(-/-) mice fed a normal or a high-cholesterol diet after short-term (ie, 18 hours) simvastatin treatment. We also studied simvastatin treatment in myocardial ischemia-reperfusion injury by subjecting apoE(-/-) mice to 30 minutes of ischemia and 24 hours of reperfusion. ApoE(-/-) mice fed a high-cholesterol diet exhibited higher blood cholesterol levels, which were not affected by short-term simvastatin treatment. However, the increased leukocyte rolling and adherence that occurred in cholesterol-fed apoE(-/-) mice (P<0.001 versus control diet) were significantly attenuated by simvastatin treatment (P<0.01 versus vehicle). Cholesterol-fed apoE(-/-) mice subjected to myocardial ischemia-reperfusion also experienced increased myocardial necrosis (P<0.01 versus control diet), which was significantly attenuated by simvastatin (P<0.01 versus vehicle). Simvastatin therapy also significantly increased vascular nitric oxide production in apoE(-/-) mice. CONCLUSIONS: Simvastatin attenuates leukocyte-endothelial cell interactions and ameliorates ischemic injury in hypercholesterolemic mice independently of lipid-lowering actions.

Low doses of superoxide dismutase and a stable prostacyclin analogue protect in myocardial ischemia and reperfusion.

The effects of low dose human superoxide dismutase and low dose taprostene, a stable analogue of prostacyclin, were investigated separately and together in a model of myocardial ischemia (1.5 h) with reperfusion (4.5 h) in open chest, anesthetized cats. Taprostene (60 ng/kg per min), human superoxide dismutase (0.25 mg/kg per h), both agents together, or their vehicle, were infused intravenously in cats starting 0.5 h after occlusion of the left anterior descending coronary artery. Neither low dose taprostene nor low dose human superoxide dismutase exerted any endothelial or myocardial protection in this model. However, the two agents together showed a significant endothelial and myocardial protection in cats with myocardial ischemia and reperfusion. Compared with cats that were untreated or received only taprostene or human superoxide dismutase, cats receiving both agents exhibited a lower plasma creatine kinase activity at every time point observed after reperfusion, a reduced area of cardiac necrosis (7 +/- 2% vs. 21 +/- 5% area at risk, p less than 0.001), lower myeloperoxidase activity in the ischemic region (p less than 0.01) and a significant preservation of vasorelaxant responses of left anterior descending coronary rings to endothelium-dependent vasodilators, acetylcholine (p less than 0.001) and A-23187 (p less than 0.001). Taprostene appears to act additively with human superoxide dismutase to inhibit neutrophil adherence and activation and to inactivate superoxide radicals, and thus reduce cellular injury 4.5 h after reperfusion of the ischemic heart. Use of this agent may allow low doses of superoxide dismutase to be used more effectively in early myocardial ischemia.

Potentiation of coronary vascular platelet adhesion by atrial pacing in the presence of arterial stenosis in dogs.

The effect of atrial pacing on coronary hemodynamics and platelet adhesion was evaluated in 13 dogs. In all 13 dogs, a snare was placed around the circumflex artery and tightened so that distal coronary artery pressure decreased to 60 to 70 mm Hg. In 10 dogs, atrial pacing was instituted at twice the heart rate at rest for 10 minutes. In three dogs, observation was continued for 10 minutes without pacing. In the 10 dogs undergoing pacing, heart rate increased from 90.5 +/- 32.6 to 173.5 +/- 45.8 beats/min. Aortic pressure was unchanged. Distal coronary artery pressure decreased from 70.8 +/- 7.8 to 53.2 +/- 10.0 mm Hg (p less than 0.05) and the pressure gradient across the stenosis increased from 47.6 +/- 12.7 to 61.2 +/- 9.1 mm Hg (p less than 0.05). Stenotic resistance increased from 2.5 +/- 0.8 to 3.6 +/- 2.4 mm Hg/ml X min-1, but coronary flow was unchanged. In all three control dogs, there was no change in coronary dynamics for the 10 minute period. In 8 of the 10 dogs that underwent pacing, platelet deposition was observed at the site of coronary stenosis. In contrast, in the three control dogs there was no platelet deposition. Atrial pacing in the presence of coronary stenosis appears to alter coronary hemodynamics such that there are activation and deposition of platelets at the site of stenosis. This platelet deposition may be transient or could become the nidus for subsequent platelet-related events in the coronary vessel.

Beneficial effects of tissue-type plasminogen activator in acute myocardial ischemia in cats.

Tissue-type plasminogen activator is a new thrombolytic agent that dissolves intravascular thrombi in coronary and peripheral vessels with less pronounced systemic lysis than that produced by streptokinase. Plasminogen activator was shown to induce reperfusion, and to salvage ischemic myocardium, by lysing experimentally induced coronary artery thrombi. The effect of a melanoma cell-derived tissue-type plasminogen activator was studied in cat myocardium rendered ischemic by coronary artery ligation for 2 hours and reperfused for another 4 hours. Plasminogen activator was infused at a rate of 500 IU X kg-1 X min-1 for the first 30 minutes of reperfusion. The marked increase in plasma creatine kinase activity during reperfusion was significantly lower in plasminogen activator-treated cats at 4, 5 and 6 hours, with 7.7 +/- 1.5 X 10(-3) IU X mg protein-1 (n = 8) in the plasminogen activator group versus 17.8 +/- 3.5 X 10(-3) IU X mg protein-1 (n = 7) in the vehicle group at 6 hours (mean +/- SEM). The area at risk in the two ischemic groups was not different, being 14.6 +/- 1.5 and 16.6 +/- 1.4% of total left ventricular mass for the treated and untreated groups, respectively. However, the mass of necrotic tissue determined histochemically was significantly lower in the plasminogen activator-treated group, accounting for 29.5 +/- 3.9% of the area at risk compared with 46.8 +/- 4.2% of area at risk in cats receiving only the vehicle (p less than 0.02).(ABSTRACT TRUNCATED AT 250 WORDS)

In vivo regulation of PECAM-1 activity during acute endothelial dysfunction in the rat mesenteric microvasculature.

The relationship between acute endothelial dysfunction and the extravasation of leukocytes was studied in vivo with intravital microscopy of the rat mesenteric microvasculature. Acute endothelial dysfunction of the rat mesenteric microvasculature was induced in vivo by superfusing the mesentery for 90 min with one of three different stimulating agents: NG-nitro-L-arginine methyl ester (L-NAME, 50 microM), thrombin (0.5 U/mL), or hydrogen peroxide (H2O2, 50 microM). All three agents induced a similar increase in leukocyte rolling and adherence, which was significantly greater than that observed in control rats superfused with Krebs-Henseleit solution (P < 0.01). Transendothelial migration of leukocytes into the perivascular space was also increased by superfusion with L-NAME, thrombin, or H2O2. However, there was a greater increase in the number of migrated leukocytes in the rat mesentery after L-NAME and H2O2 superfusion than that observed during thrombin superfusion. In vivo infusion of a neutralizing antibody against platelet-endothelial cell adhesion molecule-1 (PECAM-1) specifically inhibited L-NAME-induced and H2O2-induced migration of leukocytes but did not prevent extravasation of leukocytes induced by thrombin. In rat mesenteries superfused with the three different stimuli, immunohistochemical analysis of endothelial cell adhesion molecules expressed on the microvascular endothelium revealed a significant increase of ICAM-1, but not PECAM-1, endothelial cell surface expression (P < 0.01 and P > 0.05 vs. control rats, respectively). Our data confirm a key role for PECAM-1 acutely in leukocyte extravasation in vivo and indicate that the involvement of constitutively expressed PECAM-1 in leukocyte transendothelial migration is preferentially correlated to oxidative stress-related stimuli in the microvascular endothelium.

A novel lysophosphatidic acid analog, LXR-1035, inhibits leukocyte-endothelium interaction via inhibition of cell adhesion molecules.

A novel synthetic phosphorothioate analog of oleoyl lysophosphatidic acid LXR-1035 was studied for its ability to modulate leukocyte-endothelial cell interactions using intravital microscopy of the rat mesentery. Superfusion of the rat mesentery with 50 microM L-NAME elicited a significant, time-dependent increase in leukocyte rolling, adherence, and transmigration compared to control rats superfused with Krebs-Henseleit solution. However, superfusion of the rat mesentery with 300 nM LXR-1035 consistently attenuated 65-87% of the L-NAME-induced leukocyte rolling, adherence, and transmigration, without altering systemic blood pressure or mesenteric venular shear rate. Similar results were also obtained in rats subjected to 90 min of hemorrhage followed by 90 min of reperfusion. Resuscitation from hemorrhage increased significantly the number of rolling, adherent, and transmigrated leukocytes in the rat mesenteric microcirculation. However, superfusion of the rat mesentery with LXR-1035 markedly attenuated the leukocyte-endothelium interaction occurring after hemorrhage and reinfusion by 75+/-12%. Immunohistochemical localization of P-selectin expression on mesenteric venular endothelium was significantly increased after exposure to L-NAME and after hemorrhage-reinfusion, which was significantly attenuated by LXR-1035 (P<0.05). In addition, treatment of isolated rat neutrophils with 300 nM LXR-1035 significantly attenuated leukotriene B4-induced up-regulation of CD18 (P<0.05). Our data clearly demonstrate that LXR-1035 can potently inhibit the recruitment of leukocytes in the mesenteric rat microvasculature by attenuating cell-surface expression of adhesion molecules.

Time course of coronary vascular endothelial adhesion molecule expression during reperfusion of the ischemic feline myocardium.

The time course of endothelial P-selectin, ICAM-1, and E-selectin expression was studied in a feline model of myocardial ischemia and reperfusion. Cats were subjected to 90 min of myocardial ischemia followed by 0, 10, 20, 60, 150, or 270 min of reperfusion. At the end of reperfusion, the coronary vasculature was examined immunohistochemically to localize monoclonal antibodies (mAbs) PB1.3, RR1/1, and Cy1787 directed against P-selectin, ICAM-1, and E-selectin, respectively. Immunohistochemical localization for P-selectin, recognized by mAb PB1.3, was maximally expressed 20 min after reperfusion in 60 +/- 6% of coronary venules (P < 0.05 compared to non-reperfused controls), and covered 59 +/- 3% of the endothelial cell perimeter of immunostained coronary venules. Immunolocalization of mAb PB1.3 gradually declined at 60, 150, and 270 min of reperfusion. Immunohistochemical localization of mAb RR1/1 (anti-ICAM-1) in endothelial cells of coronary venules was observed to a modest extent in non-ischemic myocardium and at 10, 20, and 60 min of reperfusion, but was significantly increased following 150 and 270 min of reperfusion (P < 0.05 compared non-reperfused controls). At 270 min post-reperfusion, mAb RR1/1 was seen in 50 +/- 4% of coronary venules. Endothelial immunolocalization of mAb Cy1787 (anti-E-selectin) was only observed in 13 +/- 1 and 14 +/- 3% of coronary venules after 150 and 270 min of reperfusion, respectively, suggesting that pronounced expression of E-selectin does not occur within 270 min after reperfusion. These results demonstrate sequential expression of three major endothelial cell adherence molecules in situ following myocardial ischemia and reperfusion. The timing of endothelial cell expressed P-selectin and ICAM-1 could coordinate neutrophil trafficking during the early stages of reperfusion.

Myocardial protection by N,N,N-trimethylsphingosine in ischemia reperfusion injury is mediated by inhibition of P-selectin.

Polymorphonuclear leukocytes (PMNs) play an important role in myocardial ischemia/reperfusion (MI/R) injury. We examined the cardioprotective effects of N,N,N-trimethylsphingosine (TMS) in a murine model of MI (20 min) and R (24 h) injury in vivo, focusing on leukocyte-endothelial interactions. TMS is a synthetic N-methylated sphingosine derivative that has protein kinase C inhibitory activity and has been shown to prevent leukocyte activation. TMS (18 microgram/kg), administered intravenously 1 min prior to reperfusion, significantly attenuated myocardial necrotic injury assessed by myocardial creatine kinase loss compared with MI/R rats receiving only vehicle (P<0.001). Cardiac myeloperoxidase activity, an index of PMN accumulation in the ischemic myocardium, was also significantly attenuated by TMS compared with rats receiving vehicle (P<0.001). We further examined whether TMS can attenuate leukocyte-endothelial interaction by intravital microscopy. TMS significantly attenuated NG-nitro-L-arginine-methyl ester (L-NAME)-stimulated PMN rolling and adherence to the rat microvascular endothelium. This action of TMS appears to be mediated by reduction of P-selectin expression because immunohistochemical analysis demonstrated that TMS significantly attenuated endothelial P-selectin expression in the L-NAME-superfused rat mesenteric microvasculature. Similarly, TMS markedly attenuated rapid P-selectin expression in rat platelets stimulated with either thrombin or L-NAME assessed by flow cytometry. In conclusion, TMS seems to be an effective cardioprotective agent by inhibiting early leukocyte-endothelial interaction, thus preventing leukocyte accumulation in the ischemic reperfused myocardium.

Quantification of neutrophil migration following myocardial ischemia and reperfusion in cats and dogs.

Endothelial cell dysfunction and cardiac myocyte injury resulting from ischemia and reperfusion have been associated with accumulation of neutrophils in the myocardium. To determine whether the accumulation is related primarily to intravascular sequestration or extravascular infiltration of neutrophils during the early period of reperfusion, we morphometrically quantified the tissue distribution of neutrophils in cats and dogs. At the end of the reperfusion period, the base of the heart was cross-clamped to preserve neutrophil location at the moment of death. Point-counting methods were used to determine the distribution of neutrophils inside and outside coronary arterioles and venules (< or = 100 microns in diameter) as well as coronary capillaries 5-10 microns in diameter in 0.5-microns-thick, plastic-embedded sections. Ischemia-reperfusion resulted in a threefold increase in neutrophil number in the lumen of arterioles and venules at 60 min of reperfusion and up to a sevenfold increase at 270 min of reperfusion (P < .05) compared to time-matched control nonischemic hearts. The ratio of intravascular neutrophils in venules to arterioles was 2:1. Intracapillary neutrophils increased, but not significantly, at 60 min of reperfusion. At 270 min of reperfusion, intracapillary neutrophils increased 11-fold (P < .05). The percentage of total neutrophils that accumulated outside arterioles and venules in cat hearts was 8% at 60 min of reperfusion (not significant, NS) and 28% at 270 min of reperfusion (P < .05). In dog hearts, the percentages were 26% (NS) and 44% (P < .05), respectively. The percentage of total neutrophils that accumulated outside capillaries was < 6% in both cat and dog hearts (NS). The combination of rapid intravascular sequestration, delayed extravascular infiltration, and low incidence of neutrophil-cardiac myocyte contact in situ in these two species suggests that neutrophil-mediated cardiac myocyte injury during early reperfusion may initially depend on diffusion of inflammatory mediators and subsequently require direct contact between neutrophils and cardiac myocytes.