Transforming growth factor-beta(1) modulates oxidatively modified LDL-induced expression of adhesion molecules: role of LOX-1.

Oxidatively modified LDL (ox-LDL) activates a lectin-like receptor, LOX-1, which results in the expression of adhesion molecules on endothelial surface. We investigated the regulation of the expression of transforming growth factor-beta(1) (TGF-beta(1)) and its receptors by ox-LDL and the functional significance of this interaction with regard to adhesion molecule expression in human coronary artery endothelial cells (HCAECs). Ox-LDL, in a time- and concentration-dependent manner, upregulated the expression of all 3 subtypes (1, 2, and 3 [including endoglin]) of TGF-beta(1) receptors and decreased active TGF-beta(1) synthesis (all P<0.05 versus control and native-LDL-treated cells). Treatment of HCAECs with a monoclonal antibody to LOX-1 attenuated ox-LDL-mediated upregulation of TGF-beta(1) receptors and decrease in TGF-beta(1) synthesis (P<0.05 versus ox-LDL alone). Ox-LDL also enhanced the expression of P-selectin and ICAM-1 as well as monocyte adhesion to HCAECs (P<0.05 versus control untreated cells). Pretreatment with recombinant TGF-beta(1) attenuated the enhanced expression of adhesion molecules and monocyte adhesion to HCAECs (P<0.05 versus ox-LDL alone). Effects of recombinant TGF-beta(1) were blocked by antibody to TGF-beta(1) receptor type 2, but not by antibody to endoglin. Thus ox-LDL, via activation of LOX-1, increases the expression of TGF-beta(1) receptors and decreases TGF-beta(1) synthesis in HCAECs. Recombinant TGF-beta(1), by binding to TGF-beta(1) type 2 receptors, modulates ox-LDL-mediated expression of adhesion molecules and monocyte adhesion to HCAECs.

Structural requirements for microvascular permeability-increasing ability of peptides. Studies on analogues of a fibrinogen pentapeptide fragment.

A pentapeptide, Ala-Arg-Pro-Ala-Lys, liberated from fibrinogen during plasmin-mediated fibrinolysis, was shown earlier to increase microvascular permeability in rat and human skin. Eighteen new analogues have now been synthesized in addition to the 15 previously prepared and examined for their effect on permeability. The old concept that a tetrapeptide with basic amino acids at both ends and a proline residue adjacent to the N-terminal amino acid is essential for high activity on permeability, has now been challenged. The results obtained with several of the new analogues strengthen this concept. More interestingly, however, the third amino acid, which was found in earlier studies to be less sensitive to exchange, has now been deleted as well as duplicated with only a modest loss of activity of the peptide. The chirality of the C-terminal amino acid, most surprisingly, does not seem to be crucial for peptide activity. Slightly superpotent analogues were obtained on amidation of the C-terminus. In addition, a few naturally occurring peptides, namely tuftsin, substance P, neurotensin and bradykinin, the amino acid sequences of which all exhibit characteristic features of some of our active peptide analogues were investigated in the same test system. Tuftsin displayed a potency equal to that of the pentapeptide. The other three peptides were all highly superpotent in this assay system.

Structure-activity studies on synthetic analogs to vasoactive peptides derived from human fibrinogen.

Counterparts to two vasoactive peptides previously isolated from fibrin(ogen) degraded by plasmin (EC 3.4.21.7) were synthesized by the solid phase procedure. The synthetic undecapeptide (Ser-Gln-Leu-Gln-Lys-Val-Pro-Pro-Glu-Trp-Lys) was isolated in a homogeneous state by chromatography on Sephadex G-25 and DEAE-Sepharose CL-6B and the pentapeptide (Ala-Arg-Pro-Ala-Lys) by chromatography on BioGel P-6 and column zone electrophoresis. The effect of these two peptides and of fifteen analogs to the pentapeptide on microvascular permeability in rat skin was investigated. The two synthetic counterparts were as potent as the natural peptides. With respect to the analogs, the influence of different functional groups was first studied. This was followed by attempts to minimize the active structure, induce or relieve rigidity of the peptide back-bone or otherwise accomplish modifications by a change in chirality at critical positions. Our results show that the tetrapeptide Arg-Pro-Ala-Lys has the same effect on microvascular permeability as the pentapeptide in the assay system used. Basic amino acids at both ends, as well as a proline residue adjacent to the N-terminal amino acid appear important for full or essentially full activity. On the other hand, substitution of the Ala at position 4 with several other amino acids did not result in a significant loss in biological potency.

Relationship of Chlamydia pneumoniae infection to severity of human coronary atherosclerosis.

BACKGROUND: Infection with Chlamydia pneumoniae has been postulated to play a pathogenic role in atherosclerosis. We examined the role of infection with C pneumoniae in relation to the extent of coronary atherosclerosis. METHODS AND RESULTS: Coronary atherosclerosis was graded microscopically on a postmortem basis in a blinded fashion in 60 subjects as mild (n=18) or severe (n=42) atherosclerosis. Serum antibodies to C pneumoniae were measured by microimmunofluorescence test. Paraffin-embedded coronary artery specimens were examined for the presence of chlamydia by use of a genus-specific direct immunofluorescence monoclonal antibody. Frozen coronary artery specimens were examined by immunoperoxidase for the presence of C pneumoniae by use of a specific monoclonal antibody RR-402. Direct immunofluorescence was reactive in 86% of cases with severe atherosclerosis but in only 6% of cases with mild atherosclerosis (P<0.01), whereas immunoperoxidase staining was reactive in 80% and 38% of cases with severe and mild atherosclerosis, respectively (P<0. 01). Elevated IgG and IgA levels against C pneumoniae were not different in cases with severe and mild atherosclerosis (61% and 30% for severe atherosclerosis and 67% and 42% for mild atherosclerosis, respectively). CONCLUSIONS: This study supports the hypothesis that intracellular infection with C pneumoniae may relate to the severity of atherosclerosis in some subjects. Serum antibody titers against C pneumoniae do not differentiate between severe and mild atherosclerosis.

Oxidized LDL upregulates angiotensin II type 1 receptor expression in cultured human coronary artery endothelial cells: the potential role of transcription factor NF-kappaB.

BACKGROUND: We demonstrated earlier that angiotensin II (Ang II), by AT(1) receptor activation, upregulates oxidized LDL (ox-LDL) endothelial receptor LOX-1 gene expression and uptake of ox-LDL in human coronary artery endothelial cells (HCAECs). In this study, we investigated the regulation of Ang II receptors (AT1R and AT2R) by ox-LDL and the role of the redox-sensitive transcription factor NF-kappaB in this process. METHODS AND RESULTS: HCAECs were incubated with ox-LDL for 24 hours. Ox-LDL (10 to 40 microg protein/mL) upregulated AT1R but not AT2R, mRNA, or protein. Ox-LDL degraded IkappaBalpha in cytoplasm and activated transcription factor NF-kappaB (P65) in HCAEC nuclear extract. Treatment of cells with the antioxidant alpha-tocopherol (10 to 50 micromol/L) attenuated ox-LDL-mediated degradation of IkappaBalpha and activation of NF-kappaB (P65) and inhibited the upregulation of AT1R mRNA and protein. The role of NF-kappaB signal transduction was further examined by use of an NF-kappaB inhibitor, caffeic acid phenethyl ester (CAPE). Pretreatment of cells with CAPE inhibited ox-LDL-mediated degradation of IkappaBalpha and NF-kappaB activation and inhibited ox-LDL-induced upregulation of AT1R expression. Incubation of cells with both ox-LDL and Ang II increased cell injury, measured as cell viability and LDH release, compared with either ox-LDL or Ang II alone. alpha-Tocopherol as well as the specific AT1R blocker CV11974 (candesartan) attenuated the cell-injurious effects of ox-LDL. CONCLUSIONS: These observations suggest an important role of ox-LDL-mediated AT1R upregulation in cell injury. In this process, NF-kappaB activation seems to play a critical role in signal transduction. These findings provide a basis for the use of antioxidants and AT1R blockers in designing therapy of atherosclerosis.

Plasma tissue plasminogen activator inhibitor levels in coronary artery disease: correlation with age and serum triglyceride concentrations.

Increased levels of an endogenous inhibitor of tissue-plasminogen activator (t-PA) have been thought to relate to the genesis of acute myocardial ischemia. To examine the role of the rapid inhibitor of t-PA, plasma samples were analyzed from 75 patients with chest pain syndrome undergoing coronary angiography (mean age 57 years), 24 patients with clinically documented coronary artery disease (unstable angina, positive exercise stress test or previous history of myocardial infarction; mean age 58 years) and 15 young normal subjects (mean age 26 years). Plasma t-PA inhibitor levels were similar in age-matched patients regardless of the absence or presence (and degree) of coronary artery disease. Plasma t-PA inhibitor levels correlated significantly with age (r - 0.46, p less than 0.005), suggesting an age-dependent decrease in fibrinolytic activity. Plasma t-PA inhibitor levels also correlated significantly with serum triglyceride levels (r - 0.60, p less than 0.001), but not with coronary risk factors such as serum cholesterol, diabetes, hypertension, serum uric acid levels or body weight. Association of high levels of inhibitor of t-PA with hypertriglyceridemia may be of importance in the development of coronary thrombosis, especially in elderly patients. Nonetheless, this study does not suggest a pathogenic role of t-PA inhibitor in coronary atherosclerosis.

Differential effects of alpha- and gamma-tocopherol on low-density lipoprotein oxidation, superoxide activity, platelet aggregation and arterial thrombogenesis.

OBJECTIVES: This study was designed to examine the differential effects of alpha- and gamma-tocopherol on parameters of oxidation-antioxidation and thrombogenesis. BACKGROUND: Experimental studies have shown that antioxidants, such as vitamin E (alpha-tocopherol), improve atherosclerotic plaque stability and vasomotor function, and decrease platelet aggregation and tendency to thrombus formation. METHODS: Sprague Dawley rats were fed chow mixed with alpha- or gamma-tocopherol (100 mg/kg/day) for 10 days. A filter soaked in 29% FeCl3 was applied around the abdominal aorta to study the patterns of arterial thrombosis. The aortic blood flow was observed and continuously recorded using an ultrasonic Doppler flow probe. ADP-induced platelet aggregation, low-density lipoprotein oxidation induced by phorbol 12-myristate 13-acetate (PMA)-stimulated leukocytes, superoxide anion generation and superoxide dismutase (SOD) activity were also measured. RESULTS: Both alpha- and gamma-tocopherol decreased platelet aggregation and delayed time to occlusive thrombus (all p < 0.05 vs. control). Both alpha- and gamma-tocopherol decreased arterial superoxide anion generation, lipid peroxidation and LDL oxidation (all p < 0.05 vs. control), and increased endogenous SOD activity (p < 0.05). The effects of gamma-tocopherol were more potent than those of alpha-tocopherol (p < 0.05). CONCLUSIONS: This study indicates that both alpha- and gamma-tocopherol decrease platelet aggregation and delay intraarterial thrombus formation, perhaps by an increase in endogenous antioxidant activity. Gamma-tocopherol is significantly more potent than alpha-tocopherol in these effects.

Interactive role of infection, inflammation and traditional risk factors in atherosclerosis and coronary artery disease.

Although first suggested at the turn of the 20th century, there is a renewed interest in the infectious theory of atherosclerosis. Studies done in many laboratories around the world over the past several years have shown an association between markers of inflammation and coronary atherosclerosis with an exacerbation of the inflammatory process during acute myocardial ischemia, particularly in the early stages of reperfusion. It is also being recognized that the traditional risk factors, such as smoking, dyslipidemia, hypertension and diabetes mellitus, do not explain the presence of coronary atherosclerosis in a large proportion of patients. We believe that in certain genetically susceptible people, infection with very common organisms, such as Chlamydia pneumoniae or cytomegalovirus, may lead to a localized infection and a chronic inflammatory reaction. Persistence of infection may relate to the degree of inflammation and severity of atherosclerosis. Early trials with appropriate antibiotic agents in some patients with a recent history of acute myocardial infarction have led to very salutary results. If patients with an infectious basis of atherosclerosis can be identified, a therapy directed at eradication of the offending organism may be appropriate.

Concurrent nitroglycerin administration decreases thrombolytic potential of tissue-type plasminogen activator.

Dynamic coronary vasoconstriction may play a role in coronary artery reocclusion after successful thrombolysis. The effect of nitroglycerin on the thrombolytic effects of recombinant tissue-type plasminogen activator (rt-PA) was examined in dogs with an electrically induced occlusive coronary artery thrombus. Eleven dogs were randomly given rt-PA alone and seven rt-PA with nitroglycerin. The dose of rt-PA was 0.75 mg/kg body weight given over 20 min and the dose of nitroglycerin was 125 micrograms/min for 40 min. The reperfusion rate in the dogs given rt-PA alone was 73% (8 of 11 dogs) and that in the rt-PA plus nitroglycerin group was 57% (four of seven dogs) (p = NS). The time to thrombolysis (or reperfusion) in dogs receiving rt-PA plus nitroglycerin was 70% greater than in those receiving rt-PA alone (means +/- SD/29.8 +/- 9.9 versus 17.6 +/- 5.9 min, p less than 0.02), and the duration of reperfusion much shorter (11 +/- 17 versus 42 +/- 16 min, p less than 0.02). Peak coronary blood flow after reperfusion in dogs receiving rt-PA plus nitroglycerin was also less than in those receiving rt-PA alone (36 +/- 52 versus 63 +/- 20 ml/min, p less than 0.02). Reocclusion occurred in all dogs given rt-PA with nitroglycerin and in six of eight given rt-PA alone (p = NS). Plasma concentrations of rt-PA were lower when nitroglycerin was given with rt-PA alone (427 +/- 279 versus 1,471 +/- 600 ng/ml, p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Preservation of endogenous antioxidant activity and inhibition of lipid peroxidation as common mechanisms of antiatherosclerotic effects of vitamin E, lovastatin and amlodipine.

OBJECTIVES: We sought to document the common mechanisms of the antiatherogenic effects of the cholesterol-lowering hydroxy-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor lovastatin, the dihydropyridine Ca2+ blocker amlodipine and the antioxidant vitamin E. BACKGROUND: Vitamin E, HMG-CoA reductase inhibitors and Ca2+ blockers each inhibit atherosclerosis in hypercholesterolemic animals. METHODS: New Zealand White rabbits were fed regular chow (Group A), chow with 1% cholesterol (Group B), 1% cholesterol diet plus lovastatin (Group C), 1% cholesterol diet plus vitamin E (Group D) or 1% cholesterol diet plus amlodipine (Group E) for 12 weeks. The extent of aortic atherosclerosis was measured by planimetry of the sudanophilic area. Malondialdehyde (MDA) and superoxide dismutase (SOD) in blood were measured as indexes of lipid peroxidation and antioxidant activity, respectively. RESULTS: Group A rabbits showed no atherosclerosis, whereas Group B rabbits had 17.4 +/- 9.3% (mean +/- SD) of the aorta covered with atherosclerosis, and Groups C, D and E rabbits had significantly less atherosclerosis. Plasma SOD activity was lower in Group B than in Group A (6.9 +/- 1.1 vs. 12.8 +/- 1.5 U/ml, p < 0.01) and was preserved in the groups given lovastatin, vitamin E or amlodipine with a high cholesterol diet. The serum MDA level was higher in Group B rabbits than Group A rabbits (12.1 +/- 2.6 vs. 1.2 +/- 0.1 nmol/ml, p < 0.01) and increased minimally in rabbits given lovastatin, vitamin E or amlodipine with a high cholesterol diet. In in vitro experiments, both lovastatin and amlodipine preserved SOD activity and reduced the oxidizability of low density lipoproteins by rabbit leukocytes. CONCLUSIONS: This study suggests that a reduction in lipid peroxidation and preservation of SOD may be common mechanisms of antiatherosclerotic effects of lovastatin, vitamin E and amlodipine.

Increased neutrophil elastase release in unstable angina pectoris and acute myocardial infarction.

Neutrophils, a source of proteolytic enzymes and oxygen free radicals, have been shown to participate in animal models of myocardial ischemic injury. To characterize neutrophil activation in human ischemic heart disease, a specific neutrophil elastase-derived fibrinopeptide in plasma was measured in 25 patients with stable angina pectoris, 29 patients with unstable angina pectoris, 17 patients with acute myocardial infarction and 22 control subjects. Mean plasma levels (+/- standard error) of a neutrophil elastase-derived fibrinopeptide (B beta 30-43) measured by a specific radioimmunoassay were fivefold higher in patients with acute myocardial infarction (877 +/- 337 pmol/liter, p less than 0.02) and 13-fold higher in patients with unstable angina (2,277 +/- 613 pmol/liter, p less than 0.006) as compared with control subjects (172 +/- 74 pmol/liter). Mean plasma levels of peptide B beta 30-43 in patients with stable angina (676 +/- 334 pmol/liter), although higher than in control subjects, were not significantly increased (p = 0.64). Total leukocyte counts were 11.0 +/- 0.6 x 10(6)/ml in those with acute myocardial infarction, 9.2 +/- 0.7 x 10(6)/ml in those with unstable angina, 7.1 +/- 0.3 x 10(6)/ml in those with stable angina and 7.7 +/- 0.4 x 10(6)/ml in control subjects. Although total leukocyte counts in patients with unstable angina pectoris and acute myocardial infarction were higher (p less than 0.01) than in patients with stable angina or in control subjects, elevations in peptide B beta 30-43 levels were independent of the differences in both leukocyte count and absolute neutrophil count as well as in history of smoking, hypertension, diabetes mellitus or treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Recombinant lys-plasminogen, but not glu-plasminogen, improves recombinant tissue-type plasminogen activator-induced coronary thrombolysis in dogs.

OBJECTIVES: This study examined the modification of recombinant tissue-type plasminogen activator (rt-PA)-induced thrombolysis by recombinant lys-plasminogen. BACKGROUND: Recombinant tissue-type plasminogen activator restores flow in the thrombosed coronary artery, but the artery often reoccludes. The rt-PA-induced thrombolysis is a result of activation of plasminogen bound to fibrin in the thrombus and results in generation of the fibrinolytic enzyme plasmin. Small amounts of lys-plasminogen are formed when rt-PA is used. Lys-plasminogen binds to fibrin with a 10-fold greater affinity than the predominant native glu-plasminogen, leading to a loose fibrin structure. METHODS: Dogs with electrically induced occlusive intracoronary thrombus were treated with saline solution (n = 9), glu-plasminogen (2 mg/kg body weight, n = 5) or lys-plasminogen (2 mg/kg, n = 5), followed by infusion of rt-PA (1 mg/kg over 20 min) 10 min later. RESULTS: Reperfusion rates were similar in all groups of dogs, but the time to reflow was lowest in dogs given lys-plasminogen compared with those given saline solution or glu-plasminogen before rt-PA (mean [+/- SE] 14 +/- 2 vs. 22 +/- 2 and 23 +/- 3 min, respectively, p < 0.05). None of the reperfused coronary arteries reoccluded in the lys-plasminogen plus rt-PA group, whereas 75% reoccluded in dogs given saline solution plus rt-PA, and 50% reoccluded in those given glu-plasminogen plus rt-PA. Accordingly, duration of reflow was greater in the lys-plasminogen plus rt-PA group (> 120 vs. 39 +/- 7 and 82 +/- 21 min, respectively, p < 0.05). Plasminogen activator inhibitor-1 activity decreased during rt-PA infusion and thereafter increased in all dogs, but less so in dogs given lys-plasminogen (p < 0.05 vs. those given saline solution before rt-PA). CONCLUSIONS: Treatment with recombinant lys-plasminogen before rt-PA reduces time to reflow and sustains reflow after thrombolysis, whereas glu-plasminogen has no such effect.

Attenuation of coronary flow reserve and myocardial function after temporary subtotal coronary artery occlusion and increased myocardial oxygen demand in dogs.

OBJECTIVES: We examined whether subtotal coronary artery occlusion and reperfusion alter coronary flow reserve and regional myocardial function. BACKGROUND: Total coronary artery occlusion followed by reperfusion results in decreased coronary flow reserve and regional myocardial dysfunction. METHODS: Thirteen anesthetized dogs were subjected to subtotal occlusion of the left anterior descending coronary artery for 1 h, followed by reperfusion for 1 h. During subtotal left anterior descending occlusion, heart rate was increased by atrial pacing. After reperfusion, coronary flow reserve, indicated by reactive hyperemia, as well as coronary flow responses to acetylcholine and nitroglycerin, regional myocardial function and myocardial leukocyte accumulation were measured. RESULTS: After reperfusion, coronary flow reserve was decreased in the ischemic left anterior descending but not the nonischemic circumflex coronary artery region. Myocardial function was also depressed in the left anterior descending coronary region and did not improve on reperfusion. Histologic study showed no leukocyte infiltration in the ischemic left anterior descending coronary region. Myeloperoxidase, an index of myocardial leukocyte accumulation, was similar in the left anterior descending and circumflex coronary regions. Sensitivity of epicardial left anterior descending coronary artery rings to the thromboxane A2 analog U46,619 was enhanced, and relaxation of these rings in response to endothelium-dependent relaxants was decreased. CONCLUSIONS: Coronary flow reserve is reduced and regional myocardial function depressed after subtotal coronary artery occlusion and increased heart rate. A decreased synthesis or increased breakdown of endothelium-derived relaxing factor may be related to a decrease in coronary flow reserve. However, the reduction in coronary flow reserve appears to be unrelated to leukocyte accumulation in the reperfused region.

Sustained reflow in dogs with coronary thrombosis with K2P, a novel mutant of tissue-plasminogen activator.

Coronary artery reocclusion after thrombolysis with human recombinant tissue-type plasminogen activator (rt-PA) is related to the short half-life of this agent in plasma. K2P, a mutant of rt-PA lacking the fibronectin fingerlike, epidermal growth factor-like and first kringle domains (amino acids 6 to 173) and having the glycosylation site Asn184 mutagenized to Gln, has been produced in Chinese hamster ovary cells. In this study we compared the thrombolytic effect of K2P and rt-PA in dogs with electrically induced coronary artery thrombosis. Both agents were given intravenously in equimolar amounts over 20 min after the occlusive thrombus was stable for 30 min; dogs were monitored for 1 h after reperfusion if flow occurred. Coronary blood flow was restored by rt-PA in 6 (60%) of 10 dogs. The restored flow lasted for 49 +/- 12 min and mean flow at 60 min from the start of reperfusion was 7 +/- 3 ml/min. The reocclusion rate was 50% (three of six dogs). Flow was restored in five (100%) of five dogs by K2P. The restored blood flow lasted during the entire 1-h observation period in all but one dog and mean flow at 60 min was 49 +/- 16 ml/min (p less than 0.02 vs. flow in rt-PA-treated dogs). Restored coronary blood flow showed marked cyclic flow variations in rt-PA-treated but not in K2P-treated dogs.(ABSTRACT TRUNCATED AT 250 WORDS)

Pathological basis of failure of concurrent glyceryl trinitrate therapy to improve efficacy of tissue type plasminogen activator in coronary thrombosis.

STUDY OBJECTIVE: The aim was to examine the effect of glyceryl trinitrate, a potent coronary vasodilator, on the thrombolytic effects of tissue type plasminogen inhibitor (t-PA) in dogs with coronary thrombosis. DESIGN: The thrombus was formed by delivery of anodal direct current into the left anterior descending coronary artery. Fourteen dogs were randomly given t-PA alone (0.75 mg.kg-1 over 20 min) or t-PA with glyceryl trinitrate (125 micrograms.min-1 for 40 min). In four other dogs, glyceryl trinitrate was given after t-PA induced thrombolysis. Its effect on t-PA induced thrombolysis, in terms of reperfusion rate, time to thrombolysis, peak coronary blood flow, and reocclusion rate, was quantitated. Peripheral blood platelet counts and whole blood platelet aggregation were measured in all dogs. The thrombosed left anterior descending artery and normal circumflex coronary artery segments were examined by scanning electron microscopy at the end of the experiment. MEASUREMENTS AND MAIN RESULTS: The reperfusion rate in the t-PA plus glyceryl trinitrate (t-PA + GTN) group was 57% (4/7 dogs) and with t-PA alone, 71% (5/7 dogs). The time to thrombolysis in the t-PA + GTN group was greater than with t-PA alone, at 30(SD 10) v 18(7) min, p less than 0.02, and the duration of reperfusion much shorter, at 11(17) v 48(15) min, p less than 0.02. Peak coronary blood flow in the t-PA + GTN group following reperfusion was less compared with t-PA alone, at 36(52) v 53(18) ml.min-1, p less than 0.02. Reocclusion rates in the two groups were similar. Peripheral blood platelet counts decreased during thrombus formation in all dogs; this decrease stabilised when t-PA was given alone but not when it was given with glyceryl trinitrate [mean platelet count at the end of t-PA infusion 7.23(1.68) and 4.78(3.00) X 10(8).ml-1 respectively, p less than 0.02], suggesting continued sequestration of platelets in the intracoronary thrombus in the latter group. Whole blood platelet aggregation decreased significantly with t-PA alone, but less so with t-PA + GTN [magnitude of platelet aggregation 0.23(0.57) and 5.67(6.23) ohms, respectively, p less than 0.02], suggesting lower plasma concentrations of t-PA when given with glyceryl trinitrate. Glyceryl trinitrate given after thrombolysis induced by t-PA failed to sustain reperfusion. Scanning electron microscopy of occluded left anterior descending artery showed extensive endothelial injury and a thrombus composed of platelet--red blood cells--fibrin mesh. The reperfused left anterior descending artery showed extensive endothelial injury and residual thrombus consisting mostly of fibrin and red blood cells with some platelets. CONCLUSION: Glyceryl trinitrate given concurrently with t-PA or after t-PA induced thrombolysis does not modify the thrombolytic potential of t-PA. The potentially "detrimental" effects of glyceryl trinitrate may be due to increase in hepatic blood flow and subsequent enhanced catabolism of t-PA. Lack of any significant effect of therapeutic dosage of glyceryl trinitrate on platelet--fibrin rich thrombus may explain the absence of a salutary action of this agent. Dynamic coronary vasoconstriction does not play an important role in coronary reocclusion after initial thrombolysis.

Neutrophil elastase inhibitor ICI 200,880 protects against attenuation of coronary flow reserve and myocardial dysfunction following temporary coronary artery occlusion in the dog.

OBJECTIVE: Diminished coronary flow reserve and myocardial dysfunction following coronary artery occlusion and reperfusion have been attributed to neutrophil infiltration into the reperfused regions. Release of free radicals and elastase during reperfusion may also contribute to ischaemia-reperfusion induced changes. The aim of this study was to determine the effect of an elastase inhibitor on reperfusion induced attenuated coronary flow reserve and myocardial dysfunction. METHODS: Anaesthetised dogs were subjected to 1 h of left anterior descending coronary artery occlusion and 2 h of reperfusion. Ten minutes before reperfusion, dogs were randomly given saline or the neutrophil elastase inhibitor ICI 200,880 (10 mg.kg-1) and treatment was continued for the next 70 min. While the regional myocardial shortening fraction and coronary blood flow responses to acetylcholine and glyceryl trinitrate were attenuated following coronary reperfusion in saline treated dogs, similar reductions were not observed in the ICI 200,880 treated dogs (p < 0.01). Histopathology showed myocardial injury and extensive neutrophil infiltration in the reperfused regions in saline treated animals. In contrast, neutrophil infiltration was minimal in the ICI 200,880 treated dogs, in spite of myocardial injury. Myeloperoxidase, an index of neutrophil infiltration, was increased (p < 0.02 v control regions) in the reperfused regions in saline treated dogs, but not in the ICI 200,880 treated dogs. Flow cytometry also showed diminished neutrophil infiltration and oxidative burst in reperfused myocardium of ICI 200,880 treated (v saline treated) dogs. CONCLUSIONS: The elastase inhibitor ICI 200,880 protects against ischaemia-reperfusion induced attenuated coronary flow reserve and myocardial dysfunction, and this protective effect is associated with decreased neutrophil infiltration into the reperfused regions.

Aspirin does not potentiate effect of suboptimal dose of the thrombin inhibitor inogatran during coronary thrombolysis.

OBJECTIVES: Coronary artery often reoccludes after thrombolytic therapy with recombinant tissue-plasminogen activator (rt-PA). This reocclusion is thought to be due to in situ platelet activation mediated by thromboxane (Tx) A2 and thrombin; hence, aspirin and thrombin inhibitors are often used in patients with acute myocardial infarction. This study was designed to examine the modulation of coronary artery reocclusion by a novel low molecular weight direct thrombin inhibitor inogatran with or without aspirin. METHODS: 22 dogs with electrically-induced occlusive intracoronary thrombus were treated with saline (n = 7, group A), or high dose inogatran (0.25 mg/kg bolus followed by 0.6 mg/kg per h for 2 h, n = 5, group B), or low dose inogatran (0.125 mg/kg bolus followed by 0.3 mg/kg per h for 2 h, n = 5, group C), or aspirin+low dose inogatran (n = 5, Group D). Recombinant tissue-plasminogen activator (rt-PA) was infused for 20 min starting 2 min after the bolus in all dogs. Coronary artery blood flow was monitored for 120 min after rt-PA administration. RESULTS: Reperfusion rates were similar in all groups, but the time to reperfusion was shortest in group B dogs (18 +/- 2 min vs. 32 +/- 7 min in group A dogs, P < 0.05). Reocclusion rates were 80%, 0%, 50%, and 60% in groups A, B, C, and D dogs, respectively. The restored blood flow persisted for 19 +/- 10, > 120 min, 71 +/- 30 and 54 +/- 26 min in groups A, B, C, and D dogs, respectively. At the end of rt-PA infusion, prothrombin time (PT) and activated partial thromboplastin time (APTT) were increased 1.3-2 times the control value, and the changes in PT and APTT were similar in all groups. Thrombin generation and activity, assessed by rise in thrombin-antithrombin complex and fibrinopeptide A levels, and decrease in fibrinogen levels were most marked in group A dogs, and less so in group B, C and D dogs. CONCLUSIONS: These data show that high dose of direct thrombin inhibitor inogatran shortens time to reflow and abolishes coronary artery reocclusion. However, aspirin does not potentiate the effect of suboptimal doses of inogatran.

Fatty acid composition in total phospholipids of human coronary arteries in sudden cardiac death.

A study was made of the fatty acid composition of the total phospholipid fraction of human coronary arteries in 30 cases of sudden cardiac death due to ischaemic heart disease (aged 40 +/- 5 years, mean +/- S.D.) and in 29 controls (mostly traffic accident victims, aged 45 +/- 6 years). The coronary arteries from cases of sudden cardiac death showed more atherosclerotic lesions than those of controls (P < 0.001). The percentages of palmitic acid (16:0) and linoleic acid (18:2(n-6)) were significantly higher and the percentage of arachidonic acid (20:4(n-6)) and of all the other major polyunsaturated fatty acids, both n-6 and n-3, was significantly lower in cases of sudden cardiac death than in controls. In conclusion, this study showed increased percentages of saturated and reduced percentages of polyunsaturated fatty acids, except linoleic acid, in total phospholipids of human coronary arteries in cases of sudden cardiac death. The results suggest an impaired metabolism of linoleic acid, possibly due to a decreased delta-6-desaturase activity in the coronary artery wall in cases of sudden cardiac death.

PCR-based DNA typing of saliva on stamps and envelopes.

In forensic cases involving mail bombs, extortion, kidnapping or threatening letters, biological evidence such as the saliva used to attach the stamp and seal the envelope could be used for genetic analysis. We have developed a highly sensitive semi-nested PCR method for the HLA-DRB1 locus; suitable for the analyses of very limited amounts of DNA. When applied to a set of stamps and envelopes with saliva from control individuals, typing results were consistent with those obtained using hairs drawn from the same individuals. No interference was found due to DNA from the fingerprints of people handling the letters. The system was applied to three forensic cases with threatening letters. The first case resulted in an exclusion of the suspect. In the second case, the suspect could not be excluded (probability of identical genotype by chance > 0.01). These results demonstrate that biological evidence in cases with threatening letters is amenable to genetic typing.

Allele-specific HLA-DRB1 amplification of forensic evidence samples with mixed genotypes.

A major problem in analyzing forensic casework samples is the presence of genetic material from more than one individual in the material evidence. For instance, in sexual assault cases the evidence (vaginal swabs) usually contains a majority of vaginal epithelial cells and varying amounts of sperm cells from the perpetrator. Samples with mixed genotypes are also common among other biological evidence materials such as nail scrapes and mixed bloodstains. We have developed an allele-specific amplification system for the highly polymorphic HLA class II DRB1 locus that permits the detection of individual alleles in a sample with mixed genotypes, independent of the initial frequency of the alleles. Using a set of eight allele-specific amplification primers and typing the amplified fragments with sequence-specific probes, most of the 60 DRB1 alleles can be resolved. The method is highly specific and sensitive, with the potential for amplifying 15 copies of a particular allele in a background of 3 x 10(5) copies of other alleles. The method was successfully applied to three forensic cases, where the material evidence consisted of sperm stains on panties, nail scrapes and bloodstains on skin. Thus the DRB1 allele-specific amplification system can be employed for the unambiguous determination of the presence of individual alleles in materials suspected to contain mixed genotypes, even when the alleles of interest constitute only a small fraction of the total DNA.