Kousuke Marutsuka

Increased Plasminogen Activator Inhibitor Type 1 in Coronary Artery Atherectomy Specimens From Type 2 Diabetic Compared With Nondiabetic Patients A Potential Factor Predisposing to Thrombosis and Its Persistence

BACKGROUND Inhibition of fibrinolysis attributable to elevated concentrations of plasminogen activator inhibitor type 1 (PAI-1) in blood is associated with insulin resistance, hyperinsulinemia, and type 2 diabetes mellitus. Because we have shown that insulin can stimulate PAI-1 synthesis in vivo and because accelerated vascular disease is common in such patients as well, we hypothesized that increased PAI-1, potentially predisposing to thrombosis, acute occlusion, and accelerating atherosclerosis because of thrombus-associated mitogens, would be present in excess in atheroma from type 2 diabetic subjects. METHODS AND RESULTS Samples acquired by directional coronary atherectomy from 25 patients with type 2 diabetes and 18 patients without diabetes were characterized qualitatively histologically for cellularity and by immunohistochemistry visually and qualitatively and by quantitative image analysis for assessment of urokinase-type plasminogen activator (u-PA) and PAI-1. Patients with and without diabetes were similar with respect to demographic features and the distribution and severity of coronary artery disease. Substantially more PAI-1 and substantially less u-PA were present in the atherectomy samples from subjects with diabetes. CONCLUSIONS The disproportionate elevation of PAI-1 compared with u-PA observed in atheromatous material extracted from vessels of diabetic subjects is consistent with increased gene expression of PAI-1 in vessels as well as the known increase of PAI-1 in blood, presumably reflecting increased synthesis. The increased PAI-1 detected in the atheroma may contribute in vivo to accelerated or persistent thrombosis underlying acute occlusion and to vasculopathy exacerbated by clot-associated mitogens in the vessel wall. Because the changes were observed to be associated with insulin resistance and type 2 diabetes mellitus, they may be modifiable by reduction of insulin resistance with insulin sensitizers and stringent control of hyperglycemia.

Localization and activity of tissue factor in human aortic atherosclerotic lesions

Tissue factor (TF) is a transmembrane protein that serves as the major initiator of the blood coagulation cascade. The overexpression of TF antigen and mRNA has previously been reported in advanced atherosclerotic lesions. Recently TF procoagulant activity has also been identified in these lesions. However, localization and activity of TF in various stages of atherosclerosis have not yet been reported. We studied TF localization and its activity in three stages of the human atherosclerotic lesions (diffuse intimal thickening, fatty streak, and atheromatous plaque). The thoracic aortas were obtained from 23 autopsy cases and were examined immunohistochemically using an anti-human TF polyclonal antibody and biotinylated factor VIIa (FVIIa) as a probe to test the FVIIa-binding ability of TF. In addition, the TF-mediated activation of factor X (FX) was quantitatively assessed using a chromogenic assay. In lesions of the diffuse intimal thickening and the fatty streak, almost all of intimal smooth muscle cells (SMCs), macrophages, and endothelial cells were positive for TF. In the atheromatous plaques, TF antigen was detected extensively in the extracellular matrix as well as in the intimal cells. TF in all stages of atherosclerotic lesions had the ability to bind biotinylated FVIIa. TF activity was detected in each lesion and was more prominent in fatty streaks and atheromatous plaques than in the diffuse intimal thickening. These results indicate that active TF is expressed in the early stage of atherosclerotic lesions as well as in the advanced stage, and it contributes to the thrombotic property of human atherosclerotic lesions.

Protease-activated receptor 2 (PAR2) mediates vascular smooth muscle cell migration induced by tissue factor/factor VIIa complex

UNLABELLED Protease-activated receptor 2 (PAR2) is one of G-protein-coupled receptors able to be activated by trypsin and coagulation factor VIIa. We previously reported that tissue factor/factor VIIa (TF/FVIIa) complex was a strong chemotactic factor for cultured vascular smooth muscle cells (SMCs). The migratory response was dependent on a catalytic activity of FVIIa, and did not involve factor Xa and thrombin generation. In this study, we examined TF/FVIIa-induced SMC migration. METHODS The contribution of PAR2 to TF/FVIIa-induced vascular SMC migration was investigated using a modified Boydens chamber method, and the distribution of PARs in the human coronary arteries and cultured SMCs was also examined. RESULTS Trypsin and PAR2-activating peptide (AP; SLIGKV) stimulated SMC migration in a dose-dependent manner, of which abilities were comparable to those of TF/FVIIa complex and platelet-derived growth factor-BB, but PAR1-AP (TFLLR or SFLLR) or PAR4-AP (AYPGOV) did not elicit the migration. The antisera against PAR2-AP significantly inhibited TF/FVIIa-induced SMC migration, but that of PAR1-AP did not. In immunostaining, both intimal SMCs of the human coronary arteries and cultured SMCs showed positive reaction for PAR2-AP. CONCLUSION These results suggest that PAR2 in SMCs plays a crucial role in the cell migration induced by TF/FVIIa complex.

Proportion of fibrin and platelets differs in thrombi on ruptured and eroded coronary atherosclerotic plaques in humans

Objective: To determine the proportion of platelets and fibrin in coronary thrombi. Design: Immunohistochemical and morphometric means to examine the coronary arteries of 31 patients who died of acute myocardial infarction. Results: Fresh thrombi were detected in the feeding arteries of infarction areas in 23 cases (74%) and were associated with plaque rupture in 18 (78%) and plaque erosion in 5 (22%). An immunohistochemical study showed that the thrombi consisted of a mixture of fibrin and platelets as well as some other types of blood cells. The fibrin and platelet positive areas in the thrombi associated with plaque rupture accounted for 74 (19)% and 35 (20)% (p < 0.01) and those associated with erosion accounted for 51 (6)% and 70 (21)%, respectively, of the total areas. Areas of positive immunoreactivity for tissue factor and C reactive protein were also significantly greater in ruptured than in eroded plaques. Conclusion: These results indicate that the proportions of fibrin and of platelets differ in coronary thrombi on ruptured and eroded plaques. Higher proportions of tissue factor and C reactive protein contribute more significantly to thrombus formation on plaque rupture than on plaque erosion.

TNF-α and insulin, alone and synergistically, induce plasminogen activator inhibitor-1 expression in adipocytes

Obesity is associated with hyperinsulinemia and elevated concentrations of tumor necrosis factor-alpha (TNF-alpha) in adipose tissue. TNF-alpha has been implicated as an inducer of the synthesis of plasminogen activator inhibitor-1 (PAI-1), the primary physiological inhibitor of fibrinolysis, mediated by plasminogen activators in cultured adipocytes. To identify mechanism(s) through which TNF-alpha induces PAI-1, 3T3-L1 preadipocytes were differentiated into adipocytes and exposed to TNF-alpha for 24 h. TNF-alpha selectively increased the synthesis of PAI-1 without increasing activity of plasminogen activators. Both superoxide (generated by xanthine oxidase plus hypoxanthine) and hydrogen peroxide were potent inducers of PAI-1, and hydroxyl radical scavengers completely abolished the TNF-alpha induction of PAI-1. Exposure of adipocytes to TNF-alpha or insulin alone over 5 days increased PAI-1 production. These agonists exert synergistic effects. Results obtained suggest that TNF-alpha stimulates PAI-1 production by adipocytes, an effect potentiated by insulin, and that adipocyte generation of reactive oxygen centered radicals mediates the induction of PAI-1 production by TNF-alpha. Because induction of PAI-1 by TNF-alpha is potentiated synergistically by insulin, both agonists appear likely to contribute to the impairment of fibrinolytic system capacity typical in obese, hyperinsulinemic patients.

Novel distribution of adrenomedullin-immunoreactive cells in human tissues

Abstract Adrenomedullin (AM) is a novel hypotensive and vasodilator peptide. We previously examined the localization of AM in human, rat, and porcine tissues using a polyclonal antibody against synthetic human AM[40–52]. We demonstrated that AM is widely distributed in the endocrine and neuroendocrine systems, but not in the heart, kidney, or blood vessels, although high levels of AM mRNA were detected in the latter tissues. In this study, we further investigated the distribution of AM by using two newly developed monoclonal antibodies against synthetic human AM peptides, [12–25] and [46–52]. AM immunoreactivity was observed in cardiac myocytes, vascular smooth muscle cells, endothelial cells, and renal distal and collecting tubules. In addition, AM-immunoreactive (IR) cells were found in mucosal and glandular epithelia of the digestive, respiratory, and reproductive systems, as well as the endocrine and neuroendocrine systems. These findings indicate that AM-IR cells are more widely distributed in human tissues and suggest that AM might play multiple biological roles in humans.

TET2 is essential for survival and hematopoietic stem cell homeostasis.

Ten-Eleven-Translocation 2 (TET2) is an enzyme that catalyzes the conversion of 5-methylcytosine into 5-hydroxymethylcytosine (5-hmC) and thereby alters the epigenetic state of DNA; somatic loss-of-function mutations of TET2 are frequently observed in patients with diverse myeloid malignancies. To study the function of TET2 in vivo, we analyzed Ayu17-449 (TET2trap) mice, in which a gene trap insertion in intron 2 of TET2 reduces TET2 mRNA levels to about 20% of that found in wild-type (WT) mice. TET2trap/trap mice were born at Mendelian frequency but died at a high rate by postnatal day 3, indicating the essential role of TET2 for survival. Loss of TET2 results in an increase in the number of hematopoietic stem cells (HSCs)/progenitors in the fetal liver, and TET2trap/trap HSCs exhibit an increased self-renewal ability in vivo. In competitive transplantation assays, TET2trap/trap HSCs possess a competitive growth advantage over WT HSCs. These data indicate that TET2 has a critical role in survival and HSC homeostasis.

Lymphomatoid gastropathy: a distinct clinicopathologic entity of self-limited pseudomalignant NK-cell proliferation

Diagnostic errors in distinguishing between malignant and reactive processes can cause serious clinical consequences. We report 10 cases of unrecognized self-limited natural killer-cell proliferation in the stomach, designated as lymphomatoid gastropathy (LyGa). This study included 5 men and 5 women (age, 46-75 years) without any gastric symptoms. Gastroscopy showed elevated lesion(s) (diameter, ∼ 1 cm). Histologically, medium-sized to large atypical cells diffusely infiltrated the lamina propria and, occasionally, the glandular epithelium. The cells were CD2(+/-), sCD3(-), cCD3(+), CD4(-), CD5(-), CD7(+), CD8(-), CD16(-), CD20(-), CD45(+), CD56(+), CD117(-), CD158a(-), CD161(-), T cell-restricted intracellular antigen-1(+), granzyme B(+), perforin(+), Epstein-Barr early RNA(-), T-cell receptor αβ(-), and T-cell receptor γδ(-). Analysis of the 16 specimens biopsied from 10 patients led to a diagnosis of lymphoma or suspected lymphoma in 11 specimens, gastritis for 1 specimen, adenocarcinoma for 1 specimen, and LyGa or suspected LyGa for 3 specimens. Most lesions underwent self-regression. Three cases relapsed, but none of the patients died. According to conventional histopathologic criteria, LyGa is probably diagnosed as lymphoma, especially as extranodal natural killer/T-cell lymphoma, nasal type. However, LyGa is recognized as a pseudomalignant process because of its clinical characteristics. The concept of LyGa should be well recognized.

Adrenomedullin Administration Immediately After Myocardial Infarction Ameliorates Progression of Heart Failure in Rats

Background—Adrenomedullin (AM) is expressed in cardiac tissue, and plasma AM levels increase in patients with acute myocardial infarction (MI). This study was performed to determine whether AM administration immediately after acute MI inhibits progression of heart failure in rats. Methods and Results—Rats were infused with 1.0 &mgr;g/h IP AM or saline over 7 days immediately after MI inducted by left coronary ligation and were examined 9 weeks after MI. Compared with the saline infusion, AM infusion significantly improved survival (59% versus 81%; P<0.05) and body weight gain (32%; P<0.01) and reduced heart weight (−28%; P<0.01), lung weight (−26%; P<0.01), left ventricular (LV) end-diastolic pressure (11.4±2.0 versus 4.0±0.6 mm Hg, mean± SEM; P<0.01), collagen volume fraction of noninfarcted LV (−39%; P<0.05), and plasma levels of endogenous rat AM (−38%; P<0.05) without affecting infarct size. To investigate the mechanism of AM actions, another series of MI rats infused with AM were killed on day 7. AM infusion had no effect on organ weights and hemodynamic parameters on day 7 of MI but significantly reduced urinary excretion of isoprostane (−61%; P<0.01) and noninfarcted LV mRNA levels of ACE (−31%; P<0.05) and p22-phox (−30%; P<0.05). Conclusions—AM administration during the early period of MI improved the survival and ameliorated progression of LV remodeling and heart failure. This beneficial effect was accompanied by reductions in oxidative stress and ACE mRNA expression in noninfarcted LV in the AM infusion period.

Combination of a Brief Irrigation With Tissue Factor Pathway Inhibitor (TFPI) and Adenovirus-Mediated Local TFPI Gene Transfer Additively Reduces Neointima Formation in Balloon-Injured Rabbit Carotid Arteries

Background —Tissue factor pathway inhibitor (TFPI) is a physiological antagonist of TF. We tested whether a brief irrigation with TFPI protein (rTFPI) or TFPI gene transfer into injured arteries would suppress TF activity and reduce fibroproliferative changes and investigated whether a combination of these methods would show an additive effect. Methods and Results —We prepared adenoviruses expressing either TFPI (AdTFPI) or bacterial &bgr;-galactosidase (AdLacZ). Rabbit carotid arteries were balloon-injured and either infected with AdTFPI (or AdLacZ) or irrigated briefly with rTFPI (or saline). After injury, TF activity in arteries increased and was sustained; however, it was suppressed during the initial 24 hours by rTFPI irrigation (but not by gene transfer) and for a substantial period of time by TFPI gene transfer (but not by rTFPI irrigation). Four weeks later, the ratio of the intimal to medial areas was 34.3±8.7% (mean±SD, n=14) in saline-treated arteries and 33.3±4.2% in AdLacZ-infected arteries (P =NS versus saline). However, it was reduced to 25.5±8.5% in rTFPI-irrigated arteries (P <0.01 versus saline) and to 20.7±5.3% in AdTFPI-infected arteries (P <0.01 versus AdLacZ). With a combination of irrigation and gene transfer, the ratio was further reduced to 12.6±4.7% (P <0.01 versus rTFPI, P <0.05 versus AdTFPI). Systemic coagulation status was not affected in these animals. Conclusions —A combination of rTFPI irrigation and TFPI gene transfer overcomes the shortcomings shown by each method when used alone and achieves a full coverage of TF activity suppression, thereby enhancing their therapeutic effects without systemic side effects. This combination may be an effective strategy for the prevention of thrombosis and proliferative changes after angioplasty in humans.