Generation of soluble P- and E-selectins in vivo is dependent on expression of P-selectin glycoprotein ligand-1.

BACKGROUND: Factors contributing to the generation of soluble P- and E-selectins remain unclear. RESULTS: This work demonstrates that mice lacking P-selectin glycoprotein ligand-1 (Psgl-1(-/-)) are deficient in soluble P-selectin (sP-sel), which is due to a defective binding interaction between PSGL-1 and P-sel, because mice lacking alpha(1,3)-fucosyltransferase-VII are also deficient in sP-sel. Psgl-1(-/-) mice are also deficient in soluble E-selectin (sE-sel) indicating that leukocyte interactions with endothelial cells lead to the generation of sE-sel. The generation of sE-sel requires an interaction between PSGL-1 and P-sel, as deficiency of sE-sel is observed in both Psgl-1(-/-) and P-sel(-/-) mice. Bone marrow transplantation from Psgl-1(-/-) to Psgl-1(+/+) mice leads to deficiency of sP-sel and sE-sel in recipient mice, establishing the importance of bone marrow-derived PSGL-1 toward the generation of sP-sel and sE-sel. Bone marrow transplantation from P-sel(-/-) to P-sel1(+/+) mice does not lead to a significant reduction in sP-sel, confirming the importance of the endothelium toward the liberation of sP-sel. CONCLUSION: sP-sel and sE-sel reflect an interaction between leukocyte PSGL-1 and endothelial P-sel.

Fuc-TVII is required for T helper 1 and T cytotoxic 1 lymphocyte selectin ligand expression and recruitment in inflammation, and together with Fuc-TIV regulates naive T cell trafficking to lymph nodes.

To determine how the alpha(1,3)fucosyltransferases Fuc-TIV and Fuc-TVII, and the selectin ligands they control may contribute to the adaptive immune response, contact hypersensitivity (CHS) was characterized in mice deficient in either or both enzymes. We find a substantial CHS deficiency in Fuc-TVII(-/-) mice, and a complete deficiency in Fuc-TIV(-/-)/Fuc-TVII(-/-) mice. These defects are not accounted for by alterations in the number or function of epidermal Langerhans cells required for cutaneous antigen processing and presentation. By contrast, defective CHS in Fuc-TVII(-/-) mice or Fuc-TIV(-/-)/Fuc-TVII(-/-) mice is attributed in part to prominent, or nearly complete deficiencies, respectively, in the complement of naive T lymphocytes available in lymph nodes for antigen-dependent activation, expansion, differentiation, and dissemination. Fuc-TVII deficiency also deletes expression of E- and P-selectin ligands by Th1 and T cytotoxic 1 (Tc1) lymphocytes, annuls T cell trafficking to inflamed cutaneous sites in vivo, and thereby controls an essential component of the efferent phase of the cutaneous immune response. These observations indicate that collaborative contributions of Fuc-TIV and Fuc-TVII to L-selectin ligand synthesis, and to lymphocyte recruitment, are requisite components of the primary cellular immune response, and assign an essential role to Fuc-TVII in control of E- and P-selectin ligand expression by Th1 and Tc1 lymphocytes.

The alpha(1,3)fucosyltransferases FucT-IV and FucT-VII exert collaborative control over selectin-dependent leukocyte recruitment and lymphocyte homing.

E-, P-, and L-selectin counterreceptor activities, leukocyte trafficking, and lymphocyte homing are controlled prominently but incompletely by alpha(1,3)fucosyltransferase FucT-VII-dependent fucosylation. Molecular determinants for FucT-VII-independent leukocyte trafficking are not defined, and evidence for contributions by or requirements for other FucTs in leukocyte recruitment is contradictory and incomplete. We show here that inflammation-dependent leukocyte recruitment retained in FucT-VII deficiency is extinguished in FucT-IV(-/-)/FucT-VII(-/-) mice. Double deficiency yields an extreme leukocytosis characterized by decreased neutrophil turnover and increased neutrophil production. FucT-IV also contributes to HEV-born L-selectin ligands, since lymphocyte homing retained in FucT-VII(-/-) mice is revoked in FucT-IV(-/-)/FucT-VII(-/-) mice. These observations reveal essential FucT-IV-dependent contributions to E-, P-, and L-selectin ligand synthesis and to the control of leukocyte recruitment and lymphocyte homing.

Preconditioning reduces tissue complement gene expression in the rabbit isolated heart.

Both preconditioning and inhibition of complement activation have been shown to ameliorate myocardial ischemia-reperfusion injury. The recent demonstration that myocardial tissue expresses complement components led us to investigate whether preconditioning affects complement expression in the isolated heart. Hearts from New Zealand White rabbits were exposed to either two rounds of 5 min global ischemia followed by 10 min reperfusion (ischemic preconditioning) or 10 microM of the ATP-dependent K+ (KATP) channel opener pinacidil for 30 min (chemical preconditioning) before induction of 30 min global ischemia followed by 60 min of reperfusion. Both ischemic and chemical preconditioning significantly (P < 0.05) reduced myocardial C1q, C1r, C3, C8, and C9 mRNA levels. Western blot and immunohistochemistry demonstrated a similar reduction in C3 and membrane attack complex protein expression. The K(ATP) channel blocker glyburide (10 microM) reversed the depression of C1q, C1r, C3, C8, and C9 mRNA expression observed in the pinacidil-treated hearts. The results suggest that reduction of local tissue complement production may be one means by which preconditioning protects the ischemic myocardium.

Increased rat cardiac allograft survival by the glycosaminoglycan pentosan polysulfate.

BACKGROUND: Modulation of the inflammatory response has proven to be of benefit in salvaging cardiac allografts at risk of irreversible injury. Pentosan polysulfate (PPS), like heparin, is a negatively charged sulfated glycosaminoglycan (GAG) that possesses anti-inflammatory properties including the ability to inhibit activation of the complement system. This study was conducted to determine the potential of PPS to prolong allograft survival in an experimental model of cardiac transplantation. MATERIALS AND METHODS: A heterotopic cardiac transplant was performed by implanting the heart from fetal Brown Norway rats into the ear pinnae of adult Lewis rats. Vehicle (saline) or PPS (30 mg/kg) was administered subcutaneously immediately after transplantation and daily thereafter (n = 6 in each group). Another GAG, heparin, was also analyzed to determine the effect of anticoagulation on transplant survival (n = 6). RESULTS: Treatment with PPS significantly (P < 0. 05) increased allograft survival time as compared to vehicle-treated animals (8.0 +/- 0.3 days vs 5.5 +/- 0.5 days). The results noted with PPS were similar to those observed in cyclosporine (10 mg/kg; n = 6)-treated animals (8.25 +/- 0.25 days). Treatment with heparin (300 U/kg/day) did not significantly prolong cardiac graft survival time, suggesting that anticoagulation is not sufficient to prolong transplant survival. Analysis of tissue histology showed diminished transplant rejection as evidenced by decreased white blood cell infiltration and cellular necrosis. CONCLUSIONS: The results of this study indicate that PPS possesses the ability to prolong cardiac transplant viability in a heterotopic cardiac transplant model, independent of its anticoagulant actions.

Overlapping functions of E- and P-selectin in neutrophil recruitment during acute inflammation.

Selectin adhesion molecules mediate leukocyte rolling on activated endothelium, a prerequisite to leukocyte accumulation at sites of inflammation. The precise role of each selectin (E-, P-, and L-) in this process is unclear and may vary depending on the particular inflammatory stimulus, vascular bed, leukocyte subset, and species; most data suggest discrete functional roles for each selectin. To define the relative roles of E- and P-selectin in mediating neutrophil accumulation in acute dermal inflammation, mice genetically deficient in E-selectin, P-selectin, or both E- and P-selectin were injected intradermally with zymosan. Luminal endothelial expression of E- and P-selectin in response to zymosan was documented in wild-type mice by intravenous administration of fluorochrome-labeled anti-E- and anti-P-selectin antibodies. In mice deficient in E- or P-selectin, neutrophil accumulation was unchanged or only subtly reduced relative to wild-type control mice. In mice deficient in both E- and P-selectin, neutrophil accumulation was significantly reduced (87% at 4 hours and 79% at 8 hours). These data demonstrate that, in this model of acute inflammation, there is considerable overlap in the functions of E- and P-selectin; loss of both selectins was required to impair neutrophil accumulation.

Complement activation and inhibition in myocardial ischemia and reperfusion injury.

The myocardial inflammatory response that occurs as a result of ischemia and reperfusion is similar to that which occurs in other tissues. Activation of the complement system is an integral part of the initiation and maintenance of any inflammatory response. It and other immune system mediators participate in the promotion of neutrophil adherence to endothelium by modulating expression of various adhesion molecules. The complement system also serves an integral role in mediating neutrophil activation, the results of which have been documented in the setting of myocardial ischemia and reperfusion. Another aspect of the complement cascade, which has received little attention with respect to the heart, is the direct effects of complement activation such as endothelial and myocardial cell cytotoxicity mediated by the membrane attack complex. It is likely that this form of tissue injury contributes significantly to myocardial reperfusion injury. Given the numerous contributions of the complement system to the generation of the inflammatory response, and to directly-mediated tissue injury, selective inhibitors of the complement system have great potential to limit reperfusion injury. This has already been demonstrated for the complement inhibitor sCR1. In the future, it is likely that any therapeutic treatment of reperfusion injury will include modulation of the effects of complement activation.

Sulfhydryl compounds, captopril, and MPG inhibit complement-mediated myocardial injury.

Factors including complement activation, neutrophil infiltration, and oxygen-derived free radicals have been implicated in the pathogenesis of myocardial tissue injury during ischemia and reperfusion. Certain sulfhydryl-containing compounds have been shown to inhibit complement activation. The sulfhydryl compounds captopril and N-(2-mercaptopropionyl)-glycine (MPG) are antioxidant compounds that previously have been shown to protect the myocardium from ischemia and reperfusion-induced damage. In this study, captopril (an angiotensin-converting-enzyme inhibitor; ACEI) and MPG, and the non-sulfhydryl compound enalaprilat (also an ACEI) were tested for their ability to protect the isolated perfused rabbit heart against complement-induced injury. Both captopril and MPG protected hearts against complement-mediated increases in left ventricular end-diastolic pressure and increases in coronary arterial perfusion pressure in a concentration-dependent manner, whereas enalaprilat was not protective. The ability of these compounds to inhibit complement activation also was tested using an in vitro complement-mediated red blood cell hemolysis assay. These findings offer additional insight as to the mechanism whereby captopril, MPG, and possibly other sulfhydryl compounds, may be acting to provide cytoprotection during myocardial ischemia and reperfusion.

High dose intravenous aspirin, not low dose intravenous or oral aspirin, inhibits thrombus formation and stabilizes blood flow in experimental coronary vascular injury.

OBJECTIVES: The purpose of this study was to assess the anti-thrombotic potential of various forms of aspirin administration. BACKGROUND: Platelet activation in response to endothelial injury has been implicated in acute coronary syndromes. METHODS: Delivering 100-microA anodal direct current to the intima of the left circumflex coronary artery in dogs at a site of moderate external stenosis provides a thrombogenic model of vascular injury. Animals were treated with aspirin (Group I, 20 mg/kg intravenously [n = 11]; Group II, 4.6 mg/kg intravenously [n = 6]; Group III, 4.6 mg/kg orally 18 h before the experiment [n = 7]) or vehicle (Group IV, control [n = 11]). RESULTS: The time required for thrombotic occlusion to occur was longer and the incidence of thrombosis was lower in Group I (Group I, 238 +/- 7 min [n = 2]; Group II, 127 +/- 25 min [n = 3]; Group III, 156 +/- 35 min [n = 6]; Group IV, 90 +/- 11 min [n = 11]) (p < 0.05). Thrombus mass was smaller in Group I (Group I, 5.0 +/- 0.8 mg; Group II, 12.2 +/- 2.6 mg; Group III, 11.6 +/- 3.9 mg; Group IV, 9.1 +/- 1.6 mg) (p < 0.05). Initial hemodynamic variables did not differ among groups. An increase in mean arterial pressure was noted for several hours after intravenous aspirin administration in Group I (99 +/- 5 to 110 +/- 4 mm Hg) (p < 0.05). Left circumflex coronary artery blood flow was stable for 5 h in Group I (Group I, 31 +/- 2 to 26 +/- 4 ml/min) but decreased in all the other groups (Group II, 26 +/- 4 to 10 +/- 5 ml/min; Group III, 27 +/- 5 to 7 +/- 7 ml/min; Group IV, 29 +/- 4 to 0 ml/min) (p < or = 0.05). The in vivo area of left ventricle perfused by the left circumflex coronary artery was not different among groups. Platelet counts were similar and did not change over the course of the protocol. Ex vivo arachidonic acid-induced platelet aggregation decreased in all groups after aspirin (p < or = 0.001). Indium-111-labeled platelet adherence to the coronary vasculature was decreased in distal vessel segments after all doses of aspirin (p < 0.05). Platelet deposition in thrombi was similar for all treatment groups. CONCLUSIONS: High dose intravenous aspirin has salutary effects. It stabilizes left circumflex coronary artery blood flow, prolongs the time to thrombosis, reduces the incidence of thrombotic occlusion, reduces thrombus mass and limits platelet adherence to sites of arterial injury. Low dose aspirin given intravenously or orally was ineffective. When persistent intracoronary thrombi precipitate unstable coronary syndromes, high dose intravenous aspirin may be useful in the acute period even though platelets continue to interact with injured vascular segments through aspirin-insensitive mechanisms.

Soluble complement receptor type 1 prevents human complement-mediated damage of the rabbit isolated heart.

The purpose of this study was to determine if recombinant human soluble CR1 (sCR1) could prevent tissue damage associated with the activation of human complement. Directly mediated human complement-dependent myocardial injury was induced in the rabbit isolated heart perfused with a Krebs-Henseleit buffer containing 6% human plasma. There were three study groups: 1) 6% heat-inactivated human plasma (control); 2) 6% normal human plasma (NHP); or 3) 6% normal human plasma + 20 nM sCR1 (NHP + sCR1). Recorded functional parameters of the control group remained stable throughout the duration of the 70-min protocol. Complement activation in hearts perfused with 6% NHP increased the diastolic pressure; decreased developed pressure; and increased coronary perfusion pressure. These alterations were accompanied by a decrease in the maximum positive and negative dP/dt. Complement activation also increased cardiac muscle lymphatic fluid flow rate. The changes were greatest between 20 and 40 min, but persisted for the duration of the protocol. sCR1 (20 nM) in the perfusate containing 6% NHP prevented the complement-mediated alterations in the systolic, developed, and coronary perfusion pressures. sCR1 prevented the decrement in the positive and negative dP/dt, and the increase in the lymphatic fluid flow rate. Values for each of these parameters in hearts perfused with 6% NHP + sCR1 were not altered from those of controls at any time point in the protocol. Ultrastructural changes were present in tissues perfused with 6% NHP along with immunohistochemical evidence for presence of the terminal C5b-9 complex. sCR1 prevented the ultrastructural changes and the formation of the terminal complex. sCR1 offers significant protection against the cytolytic effects resulting from activation of the human complement system.

Effects of complement activation in the isolated heart. Role of the terminal complement components.

The mechanisms of the complement-mediated myocardial injury associated with ischemia and reperfusion have not been elucidated fully. Complement activation may directly mediate injury through actions of the anaphylatoxins C3a and C5a or generation of the membrane attack complex C5b-9. A model was developed to examine the direct effects of complement activation on heart function, assess myocardial tissue damage, and determine which complement components mediate tissue injury. Isolated rabbit hearts were perfused with Krebs-Henseleit buffer by using a modified Langendorff apparatus. Human plasma was added to the perfusate as a source of complement. Rabbit tissue activates human complement. Treatment with 6% normal plasma resulted in complement activation as assessed by the generation of Bb, C3a, C5a, and SC5b-9. Functional changes in cardiac performance became apparent 7-15 minutes after plasma addition and developed fully over the next 20-30 minutes. The effects were dependent on the complement titer and included 1) an increase in the end-diastolic pressure, 2) a decrease in the developed pressure, 3) an increase in the coronary perfusion pressure, and 4) an increase in lymphatic fluid formation. These effects were not elicited when an inhibitor of complement activation (FUT-175) was present or when heat-inactivated plasma was used. The effects of complement activation on myocardial function could not be reproduced by treatment with recombinant human C5a, zymosan-activated plasma, or plasma selectively depleted of C8. Myocardial tissue accumulated sodium and calcium and lost potassium as a result of complement activation. Activation caused the release of creatine kinase from myocytes and an increase in the radiolabeled albumin space of the hearts. The data demonstrate that complement activation caused decrements in myocardial function and increased the coronary perfusion pressure and lymphatic fluid flow rate. The effects were not mediated by the anaphylatoxins but were dependent on the distal complement component C8, suggesting that C5b-9 was responsible for the physiological changes. Complement activation directly mediated tissue injury in a manner consistent with plasmalemmal disruption as a result of C5b-9 formation. The data suggest that the C5b-9 complex, which is known to form under conditions of ischemia, may contribute directly to myocardial cell injury.

Combined adenosine and lidocaine administration limits myocardial reperfusion injury.

The endogenous compound adenosine may play a role in limiting myocardial ischemia-reperfusion injury through its ability to cause vasodilation, modulate cardiac adrenergic responses, inhibit neutrophil function, or modulate energy supply and demand of the myocardium. The local anesthetic lidocaine has been shown to be protective against myocardial ischemia-reperfusion injury, although its mechanism of action remains unresolved. We hypothesized that administration of exogenous adenosine during reperfusion would limit the size of the infarct that results from a period of ischemia and reperfusion only when the animals are treated with lidocaine. Male, mongrel dogs (13.0-20.0 kg) were anesthetized (30 mg/kg i.v. sodium pentobarbital), and a left thoracotomy was performed. The left circumflex coronary artery (LCx) was isolated and instrumented with an electromagnetic flow probe, a 25-gauge nonobstructing intracoronary catheter, and a critical stenosis. The dogs were allocated randomly to one of four groups: 1) control, n = 13, (saline), 2) adenosine, n = 13, (0.15 mg/kg/ml/min i.c. for the first hour of reperfusion), 3) lidocaine, n = 9, (2.0 mg/kg i.v. given immediately before coronary artery occlusion and just before reperfusion), or 4) adenosine plus lidocaine, n = 11. The LCx was occluded for 90 minutes and reperfused for 6 hours. Regional myocardial blood flow (RMBF) was determined (n = 6 per group) at 80 minutes of occlusion and at 45 minutes of reperfusion with radiolabeled microspheres. RMBF determinations revealed an increase in blood flow to the inner two thirds of the myocardium at 45 minutes of reperfusion only in the presence of the combined treatment. Adenosine treatment alone or lidocaine treatment alone did not affect RMBF. Quantification of infarct size (triphenyltetrazolium method) expressed as a percent of the area at risk revealed a significant limitation of infarct size only in the group treated with both adenosine and lidocaine: control, 47.8 +/- 6.6%; adenosine, 45.0 +/- 3.2%; lidocaine, 46.9 +/- 6.0%; and adenosine and lidocaine, 20.8 +/- 5.6%. Statistical analyses were performed with two-way analysis of variance to account for the two individual drug treatments. The findings show that intracoronary administration of exogenous adenosine, at the dose used, is only effective at limiting myocardial infarct size when administered to lidocaine-treated animals.

Antiplatelet antibody [7E3 F(ab')2] prevents rethrombosis after recombinant tissue-type plasminogen activator-induced coronary artery thrombolysis in a canine model.

Coronary artery rethrombosis can complicate initially effective thrombolytic therapy. Platelets interacting with injured vascular endothelium in a region along the coronary artery with reduced luminal cross-sectional area contribute to rethrombosis. The purpose of this study was to investigate the potential of the F(ab')2 fragment of the murine monoclonal antibody 7E3 [7E3 F(ab')2] to prevent rethrombosis after intracoronary clot lysis with recombinant tissue-type plasminogen activator (rt-PA) in an experimental model. The 7E3 F(ab')2 binds to the platelet glycoprotein IIb/IIIa complex (GPIIb/IIIa), thereby preventing platelet-fibrinogen interaction and intravascular thrombus formation. Experimental coronary artery thrombosis was produced in the anesthetized dog by application of direct anodal current to the intimal surface of the left circumflex coronary artery in the region of an external stenosis. Lysis of the established intracoronary thrombus was achieved with the intravenous administration of rt-PA (25 mg) after which the animals were randomized into two groups. Group 1 (n = 10) served as the control, receiving the saline diluent, and group 2 (n = 9) received 7E3 F(ab')2, given as a single intravenous injection (0.8 mg/kg). The times required for occlusive thrombus formation, rt-PA-induced thrombolysis, and rethrombosis (if it occurred) were similar in the animals treated with saline and those treated with 7E3 F(ab')2. The initial left circumflex coronary artery blood flow was similar in both groups but decreased to a negligible level in group 1. In group 2, left circumflex coronary artery blood flow declined modestly (24 +/- 2 to 10 +/- 2 ml/min). Rethrombosis occurred in all animals in group 1 but in only two of nine animals in group 2 (p less than 0.05). Oscillations in coronary blood flow preceded rethrombosis in group 1, whereas 7E3 F(ab')2 stabilized left circumflex coronary artery blood flow patterns during the course of teh experimental protocol (5.2 +/- 0.9 vs. 0.7 +/- 0.4 oscillations, respectively; p less than 0.05). Thrombus mass recovered from the left circumflex coronary artery at the conclusion of each experiment was greater in group 1 as compared with group 2 (7.0 +/- 2.3 vs. 1.5 +/- 0.7 mg, respectively; p less than 0.05). The area of left ventricle at risk for infarction was similar in both groups but infarct size, infarction/at risk assessed histochemically, was larger in group 1 than group 2 (35 +/- 9% vs. 6 +/- 4%, respectively; p less than 0.05). Platelet aggregation induced by ADP and arachidonic acid was similar at baseline for all of the animals.(ABSTRACT TRUNCATED AT 400 WORDS)

Prostaglandin E1 hyperthermia in water or food deprived rats.

The effect of 48 hours of water deprivation on the colonic temperature response to intrahypothalamic injection of prostaglandin E1 (PGE1) was investigated in adult male rats. Water deprivation did not alter colonic temperature of rats at a neutral ambient temperature. Administration of PGE1 at doses of 50, 200 and 400 ng gave rise to a short latency dose dependent hyperthermia in both control and water deprived rats. Water deprived rats had significantly greater increases in colonic temperature following the two higher doses of PGE1. Control rats and water deprived rats exposed to the cold (5 degrees C) had decreases in colonic temperature which were not significantly different. Water deprivation, which should increase the plasma levels of the putative endogenous antipyretic vasopressin, does not attenuate PGE1 hyperthermia but has a slight enhancing effect. Following food deprivation for 48 hours rats had a slight but significantly greater increase in colonic temperature following intrahypothalamic injection of 200 ng PGE1. Thus the water deprivation induced change in responsiveness to PGE1 may be due to the decrease in food intake which accompanies water deprivation. The mechanism by which rats exhibit an enhanced febrile response to PGE1 administration following food or water deprivation is not yet known.

Interaction of the formed elements of blood with the coronary vasculature in vivo.

Considerable attention is being given to the interactions that occur among blood platelets, neutrophils, and the vascular endothelium. There is an increasing awareness that the various blood elements interact in the process of thrombus formation and vascular occlusion. In addition, interactions among these cells can lead to the formation and release of vasoactive substances that have the potential to modulate regional blood flow. This review focuses on the coronary vascular bed and an assessment of how cell-cell interactions, under normal physiological conditions as well as in the presence of myocardial injury, may lead to alterations in coronary vascular resistance and myocardial function. Should related events be operative in human clinical states of disease, the circulating elements of the blood may serve as targets in the development of therapeutic interventions to regulate myocardial blood flow.