Yunosuke Matsuura

Increased Metabolite Levels of Glycolysis and Pentose Phosphate Pathway in Rabbit Atherosclerotic Arteries and Hypoxic Macrophage

Aims Inflammation and possibly hypoxia largely affect glucose utilization in atherosclerotic arteries, which could alter many metabolic systems. However, metabolic changes in atherosclerotic plaques remain unknown. The present study aims to identify changes in metabolic systems relative to glucose uptake and hypoxia in rabbit atherosclerotic arteries and cultured macrophages. Methods Macrophage-rich or smooth muscle cell (SMC)-rich neointima was created by balloon injury in the iliac-femoral arteries of rabbits fed with a 0.5% cholesterol diet or a conventional diet. THP-1 macrophages stimulated with lipopolysaccharides (LPS) and interferon-γ (INFγ) were cultured under normoxic and hypoxic conditions. We evaluated comprehensive arterial and macrophage metabolism by performing metabolomic analyses using capillary electrophoresis-time of flight mass spectrometry. We evaluated glucose uptake and its relationship to vascular hypoxia using 18F-fluorodeoxyglucose (18F-FDG) and pimonidazole, a marker of hypoxia. Results The levels of many metabolites increased in the iliac-femoral arteries with macrophage-rich neointima, compared with those that were not injured and those with SMC-rich neointima (glycolysis, 4 of 9; pentose phosphate pathway, 4 of 6; tricarboxylic acid cycle, 4 of 6; nucleotides, 10 of 20). The uptake of 18F-FDG in arterial walls measured by autoradiography positively correlated with macrophage- and pimonidazole-immunopositive areas (r = 0.76, and r = 0.59 respectively; n = 69 for both; p<0.0001). Pimonidazole immunoreactivity was closely localized with the nuclear translocation of hypoxia inducible factor-1α and hexokinase II expression in macrophage-rich neointima. The levels of glycolytic (8 of 8) and pentose phosphate pathway (4 of 6) metabolites increased in LPS and INFγ stimulated macrophages under hypoxic but not normoxic condition. Plasminogen activator inhibitor-1 protein levels in the supernatant were closely associated with metabolic pathways in the macrophages. Conclusion Infiltrative macrophages in atherosclerotic arteries might affect metabolic systems, and hypoxia but not classical activation might augment glycolytic and pentose phosphate pathways in macrophages.

Paucity of CD34-positive cells and increased expression of high-mobility group box 1 in coronary thrombus with type 2 diabetes mellitus

To examine the presence of CD34-positive cells and intranuclear factors in acute coronary thrombi, we compared thrombi in patients with type 2 diabetes mellitus (DM, n = 21) and without DM (n = 29). Immunohistochemical staining revealed the constitutive presence of platelets, fibrin, erythrocytes, neutrophils, extracellular high-mobility group box 1 (HMGB-1), and histone H3 in all thrombi. There were significantly more oval or flat CD34-positive cells and significantly larger HMGB-1-positive areas in the thrombi from patients with DM. The flat CD34-positive cells expressed ecto-nucleoside triphosphate diphosphohydrolase (a platelet aggregation inhibitor). The number of CD34-positive cells was negatively correlated with the serum levels of glucose and hemoglobin A1c, whereas the HMGB-1-positive area was positively correlated with the levels of serum glucose. The paucity of CD34-positive cells and the high levels of HMGB-1 expression in acute coronary thrombi from patients with type 2 DM could facilitate thrombus formation.

Human C-reactive protein enhances thrombus formation after neointimal balloon injury in transgenic rabbits

Summary.  Background: High plasma levels of C‐reactive protein (CRP) constitute a powerful predictive marker of cardiovascular events. Several lines of evidence suggest that CRP has prothrombogenic effects. However, whether CRP directly participates in the pathogenesis of thrombosis in vivo has not been fully clarified. Objective: To test whether human CRP (hCRP) affects arterial thrombus formation after balloon injury of smooth muscle cell (SMC)‐rich or macrophage‐rich neointima. Methods: We compared the susceptibility of transgenic (Tg) rabbits expressing hCRP (46.21 ± 13.85 mg L−1, n = 22) and non‐Tg rabbits to arterial thrombus formation after balloon injury of SMC‐rich or macrophage‐rich neointima. Results: Thrombus size on SMC‐rich or macrophage‐rich neointima was significantly increased, and was accompanied by an increase in fibrin content in hCRP‐Tg rabbits, as compared with non‐Tg rabbits. Thrombus size did not significantly differ between SMC‐rich and macrophage‐rich neointima in hCRP‐Tg rabbits. Tissue factor (TF) mRNA expression and activity in these neointimal lesions were significantly increased in hCRP‐Tg rabbits as compared with non‐Tg rabbits. The degree of CRP deposition correlated with the elevated TF expression and thrombus size on injured neointima. In addition, hCRP isolated from hCRP‐Tg rabbit plasma induced TF mRNA expression and activity in rabbit cultured vascular SMCs. Conclusions: These results suggest that elevated plasma hCRP levels promote thrombus formation on injured SMC‐rich neointima by enhancing TF expression, but have no additive effects in macrophage‐rich neointima.

Vascular wall hypoxia promotes arterial thrombus formation via augmentation of vascular thrombogenicity

Atherosclerotic lesions represent a hypoxic milieu. However, the significance of this milieu in atherothrombosis has not been established. We aimed to assess the hypothesis that vascular wall hypoxia promotes arterial thrombus formation. We examined the relation between vascular wall hypoxia and arterial thrombus formation using a rabbit model in which arterial thrombosis was induced by 0.5 %-cholesterol diet and repeated balloon injury of femoral arteries. Vascular wall hypoxia was immunohistochemically detected by pimonidazole hydrochloride, a hypoxia marker. Rabbit neointima and THP-1 macrophages were cultured to analyse prothrombotic factor expression under hypoxic conditions (1 % O2). Prothrombotic factor expression and nuclear localisation of hypoxia-inducible factor (HIF)-1α and nuclear factor-kappa B (NF-κB) p65 were immunohistochemically assessed using human coronary atherectomy plaques. Hypoxic areas were localised in the macrophage-rich deep portion of rabbit neointima and positively correlated with the number of nuclei immunopositive for HIF-1α and NF-κB p65, and tissue factor (TF) expression. Immunopositive areas for glycoprotein IIb/IIIa and fibrin in thrombi were significantly correlated with hypoxic areas in arteries. TF and plasminogen activator inhibitor-1 (PAI-1) expression was increased in neointimal tissues and/or macrophages cultured under hypoxia, and both were suppressed by inhibitors of either HIF-1 or NF-κB. In human coronary plaques, the number of HIF-1α-immunopositive nuclei was positively correlated with that of NF-κB-immunopositive nuclei and TF-immunopositive and PAI-1-immunopositive area, and it was significantly higher in thrombotic plaques. Vascular wall hypoxia augments the thrombogenic potential of atherosclerotic plaque and thrombus formation on plaques via prothrombotic factor upregulation.

Human Coronary Thrombus Formation Is Associated With Degree of Plaque Disruption and Expression of Tissue Factor and Hexokinase II

BACKGROUND Atherosclerotic plaque thrombogenicity is a critical factor that affects thrombus formation and the onset of acute myocardial infarction (AMI). The aim of this study was to identify the vascular factors involved in thrombus formation and AMI onset. METHODSANDRESULTS Culprit lesions in 40 coronary arteries with thrombi at autopsy after lethal AMI and non-cardiac death (asymptomatic plaque disruption) were analyzed on histology. Thrombus size, ratio of thrombus to lumen area, length of plaque disruption, and immunopositive areas for tissue factor (TF) and hexokinase (HK)-II were significantly larger in coronary arteries with AMI than with asymptomatic plaque disruption. The size of coronary thrombus positively correlated with the length of plaque disruption (r=0.80) and with immunopositive areas for TF (r=0.38) and HK-II (r=0.40). Because both M1 and M2 macrophages express TF and HK-II in symptomatic plaques, we assessed TF and HK-II expression in M1- and M2-polarized macrophages. The expression of TF was increased and that of HK-II was decreased in M2-, compared with M1-polarized THP-1 macrophages. Inhibiting glycolysis enhanced TF expression in the macrophages partly via hypoxia inducible factor-1α. CONCLUSIONS The degree of plaque disruption and expression of TF and HK-II appear to be important vascular factors for AMI onset, and polarized macrophages make a distinct contribution to thrombogenicity and glucose metabolism.

Altered glucose metabolism and hypoxic response in alloxan-induced diabetic atherosclerosis in rabbits

Diabetes mellitus accelerates atherosclerosis that causes most cardiovascular events. Several metabolic pathways are considered to contribute to the development of atherosclerosis, but comprehensive metabolic alterations to atherosclerotic arterial cells remain unknown. The present study investigated metabolic changes and their relationship to vascular histopathological changes in the atherosclerotic arteries of rabbits with alloxan-induced diabetes. Diabetic atherosclerosis was induced in rabbit ilio-femoral arteries by injecting alloxan (100 mg/kg), injuring the arteries using a balloon, and feeding with a 0.5% cholesterol diet. We histologically assessed the atherosclerotic lesion development, cellular content, pimonidazole positive-hypoxic area, the nuclear localization of hypoxia-inducible factor-1α, and apoptosis. We evaluated comprehensive arterial metabolism by performing metabolomic analyses using capillary electrophoresis-time of flight mass spectrometry. We evaluated glucose uptake and its relationship to vascular hypoxia using 18F-fluorodeoxyglucose and pimonidazole. Plaque burden, macrophage content, and hypoxic areas were more prevalent in arteries with diabetic, than non-diabetic atherosclerosis. Metabolomic analyses highlighted 12 metabolites that were significantly altered between diabetic and non-diabetic atherosclerosis. A half of them were associated with glycolysis metabolites, and their levels were decreased in diabetic atherosclerosis. The uptake of glucose evaluated as 18F-fluorodeoxyglucose in atherosclerotic lesions increased according to increased macrophage content or hypoxic areas in non-diabetic, but not diabetic rabbits. Despite profound hypoxic areas, the nuclear localization of hypoxia-inducible factor-1α decreased and the number of apoptotic cells increased in diabetic atherosclerotic lesions. Altered glycolysis metabolism and an impaired response to hypoxia in atherosclerotic lesions under conditions of insulin-dependent diabetes might be involved in the development of diabetic atherosclerosis.

Elevated plasma factor VIII enhances venous thrombus formation in rabbits: Contribution of factor XI, von Willebrand factor and tissue factor

Elevated plasma levels of factor VIII (FVIII) are associated with increased risk of deep venous thrombosis. The aim of this study is to elucidate how elevated FVIII levels affect venous thrombus formation and propagation in vivo. We examined rabbit plasma FVIII activity, plasma thrombin generation, whole blood coagulation, platelet aggregation and venous wall thrombogenicity before and one hour after an intravenous infusion of recombinant human FVIII (rFVIII). Venous thrombus induced by the endothelial denudation of rabbit jugular veins was histologically assessed. Thrombus propagation was evaluated as indocyanine green fluorescence intensity. Argatroban, a thrombin inhibitor, and neutralised antibodies for tissue factor (TF), factor XI (FXI), and von Willebrand factor (VWF) were infused before or after thrombus induction to investigate their effects on venous thrombus formation or propagation. Recombinant FVIII (100 IU/kg) increased rabbit plasma FVIII activity two-fold and significantly enhanced whole blood coagulation and total plasma thrombin generation, but did not affect initial thrombin generation time, platelet aggregation and venous wall thrombogenicity. The rFVIII infusion also increased the size of venous thrombus 1 hour after thrombus induction. Argatroban and the antibodies for TF, FXI or VWF inhibited such enhanced thrombus formation and all except TF suppressed thrombus propagation. In conclusion, elevated plasma FVIII levels enhance venous thrombus formation and propagation. Excess thrombin generation by FXI and VWF-mediated FVIII recruitment appear to contribute to the growth of FVIII-driven venous thrombus.

Indoleamine 2,3-dioxygenase 1 in coronary atherosclerotic plaque enhances tissue factor expression in activated macrophages

Recent clinical studies have found that changes in the kynurenine (Kyn) pathway of tryptophan (Trp) metabolism are associated with cardiovascular events. However, the roles of the Kyn pathway on vascular wall thrombogenicity remain unknown. Indoleamine 2,3‐dioxygenase 1 (IDO1) is a rate‐limiting enzyme of the Kyn pathway.

A Baker Cyst Accompanied by Venous Thromboembolism

A 36-year-old woman who had received low-dose estrogen and progesterone therapy to alleviate menstrual cramps was referred to the emergency department of our hospital due to left calf pain with mild edema. The patient‘s laboratory data revealed elevated levels of D-dimer (6.94 μg/mL) and fibrinogen degradation product (FDP) (15.0 μg/mL). Computed tomography demonstrated the presence of a pulmonary thrombotic embolism (PTE) (Picture A) and deep vein thrombosis (DVT) in the left posterior tibial vein (Picture B). The left popliteal vein was also found to be externally compressed by a Baker cyst (BC) (Picture C). These findings were also confirmed by an ultrasound examination (Picture D, E). Symptomatic BC is sometimes associated with pseudothrombophlebitis (1) which is characterized by the absence of thrombus; however, true DVT and PTE were both present in this patient. However, it should be noted that it is difficult to distinguish the contribution of BC-induced vein compression from that of hormone therapy to the occurrence of venous thromboembolism.

SIGNIFICANCE OF CARDIAC TROPONIN T LEVELS IN SUPRAVENTRICULAR TACHYCARDIA

Background: Cardiac troponin T is sensitive and specific markers of myocardial injury and is used routinely for the diagnosis of acute coronary syndrome. Recently, the magunitude of troponin T levels in heart failure patients has been reported to correlate with severity of the disease and with adverse outcomes. They may suggest ongoing myocardial damage. In supraventricular tachycardia, common atrial flutter (AFL) and atrial tachycardia (AT) often produce changes in cardiac function and structure, but atrioventricular nodal reentrant tachycardia (AVNRT) and atrioventricular reentrant tachycardia (AVRT) do not. To our knowledge, there are no reports about the relationship between the levels of troponin T and the types of supraventricular tachycardia. We examined the clinical usefulness of previously unmeasurable levels of troponin T (hs-TnT) by using highly sensitive assay for the differential diagnosis of supraventricular tachycardia.