Administration of pigment epithelium-derived factor inhibits left ventricular remodeling and improves cardiac function in rats with acute myocardial infarction.

Oxidative stress and inflammation are involved in cardiac remodeling after acute myocardial infarction (AMI). We have found that pigment epithelium-derived factor (PEDF) inhibits vascular inflammation through its anti-oxidative properties. However, effects of PEDF on cardiac remodeling after AMI remain unknown. We investigated whether PEDF could inhibit left ventricular remodeling and improve cardiac function in rats with AMI. AMI was induced in 8-week-old Sprague-Dawley rats by ligation of the left ascending coronary artery. Rats were treated intravenously with vehicle or 10 µg PEDF/100 g b.wt. every day for up to 2 weeks after AMI. Each rat was followed until 16 weeks of age. PEDF levels in infarcted areas and serum were significantly decreased at 1 week after AMI and remained low during the observational periods. PEDF administration inhibited apoptotic cell death and oxidative stress generation around the infarcted areas at 2 and 8 weeks after AMI. Further, PEDF injection suppressed cardiac fibrosis by reducing transforming growth factor-ß and type III collagen expression, improved left ventricular ejection fraction, ameliorated diastolic dysfunction, and inhibited the increase in left ventricular mass index at 8 weeks after AMI. The present study demonstrated that PEDF could inhibit tissue remodeling and improve cardiac function in AMI rats. Substitution of PEDF may be a novel therapeutic strategy for cardiac remodeling after AMI.

Isolation and characterization of a virus (CvV-BW1) that infects symbiotic algae of Paramecium bursaria in Lake Biwa, Japan.

BACKGROUND: We performed an environmental study of viruses infecting the symbiotic single-celled algae of Paramecium bursaria (Paramecium bursaria Chlorella virus, PBCV) in Lake Biwa, the largest lake in Japan. The viruses detected were all Chlorella variabilis virus (CvV = NC64A virus). One of them, designated CvV-BW1, was subjected to further characterization. RESULTS: CvV-BW1 formed small plaques and had a linear DNA genome of 370 kb, as judged by pulsed-field gel electrophoresis. Restriction analysis indicated that CvV-BW1 DNA belongs to group H, one of the most resistant groups among CvV DNAs. Based on a phylogenetic tree constructed using the dnapol gene, CvV was classified into two clades, A and B. CvV-BW1 belonged to clade B, in contrast to all previously identified virus strains of group H that belonged to clade A. CONCLUSIONS: We conclude that CvV-BW1 composes a distinct species within C. variabilis virus.

HbA1c levels measured by enzymatic assay during off-site health checkups are lower than those measured by on-site HPLC assay.

HbA1c is widely used as a therapeutic target marker and as a diagnostic marker for diabetes mellitus. This has led to an increasing frequency of HbA1c measurements in current health checkups throughout Japan. In the present study, we compared the HbA1c levels measured by an enzymatic assay (EA-HbA1c) off-site during health checkups with the HbA1c levels measured by on-site ion-exchange high-performance liquid chromatography (HPLC; HPLC-HbA1c) in a hospital. A total of 96 individuals (53 males and 43 females; age, 68.9 ± 8.4 years old; 70 diabetic and 26 non-diabetic individuals) whose HbA1c levels were measured by both the methods listed above were included in the study. Since no HPLC-HbA1c levels were measured on the day of the health checkup, HPLC-HbA1c levels were estimated using HPLC-HbA1c levels measured before and after the health checkup. A significant correlation of HbA1c levels was observed between the two groups (R = 0.973; p < 0.001). However, EA-HbA1c levels measured off-site during health checkups are lower than estimated HPLC-HbA1c levels measured on-site (6.37 ± 0.75% vs. 6.69 ± 0.75%; p < 0.001). Since lower EA-HbA1c levels measured during health checkups, which diverged from on-site measurements, may lead to underestimating diabetes mellitus, accurate measurement of HbA1c is required irrespective of the measuring method. Further investigation of the cause of falsely low EA-HbA1c levels and the strategy for reconciling HbA1c to reflect plasma glucose accurately are warranted.

Usefulness of the SAGE score to predict elevated values of brachial-ankle pulse wave velocity in Japanese subjects with hypertension.

The score based on the office systolic blood pressure, age, fasting blood glucose level, and estimated glomerular filtration rate (SAGE score) has been proposed as a useful marker to identify elevated values of carotid-femoral pulse wave velocity (PWV). The present cross-sectional study was conducted to examine whether the SAGE score is also a useful marker to identify subjects with elevated brachial-ankle PWV values in Japanese subjects with hypertension. We measured the brachial-ankle PWV and calculated the SAGE score in a total of 1019 employees of a Japanese company with hypertension and 817 subjects with hypertension derived from a multicenter study cohort. The analyses in this study were based on data from these two study groups as well as on a composite population of the two (n = 1836). The receiver operating characteristic curve analysis showed that the area under the curve to identify subjects with brachial-ankle PWV values of ≥1800 cm/s was over 0.70 in each of the three study groups. Even after adjustments, a SAGE score ≥7 had a significant odds ratio for identifying subjects with brachial-ankle PWV values ≥1800 cm/s in the 1836 study subjects from the composite occupational and multicenter study cohort (odds ratio = 2.1, 95% confidence interval = 1.4-3.0, P < 0.01). Thus, in Japanese subjects with hypertension, the SAGE score may be a useful marker for identifying subjects with elevated brachial-ankle PWV values.

Effect of short-term colchicine treatment on endothelial function in patients with coronary artery disease.

BACKGROUND: Inflammation is associated with endothelial dysfunction and plays an important role in the pathogenesis and development of cardiovascular diseases. It has been shown that colchicine, an anti-inflammatory drug, improves the cardiovascular outcome in patients with cardiovascular disease. The purpose of this study was to evaluate the short-term effect of low-dose colchicine on endothelial function in patients with coronary artery disease (CAD). METHODS: This was a double-blind, randomized, placebo-controlled, crossover-within-subject clinical trial. A total of 28 patients with CAD received low-dose colchicine (0.5 mg/day) or a placebo for 7 days with a washout period of at least 14 days. Flow-mediated vasodilation (FMD) and serum concentrations of high-sensitivity C-reactive protein (hs-CRP) were measured after the 7-day treatment with colchicine or the placebo. RESULTS: The serum concentration of hs-CRP was significantly decreased after administration of colchicine compared with that after administration of the placebo [median (interquartile range): 0.04 (0.02-0.08) mg/dL vs. 0.07 (0.04-0.11) mg/dL, P = 0.003], while there was no significant difference in FMD between the treatments [median (interquartile range): 3.1% (1.5-5.3%) vs. 3.3% (1.9-5.2%), P = 0.384]. Colchicine, however, significantly improved FMD in coronary artery disease patients with white blood cell (WBC) counts of ≥7500 WBC/mm3 [median (interquartile range): 3.3% (2.1-6.6%) vs. 2.0% (1.4-3.8%), P = 0.043]. CONCLUSIONS: Administration of low-dose colchicine did not improve endothelial function in patients with CAD, but exploratory analysis suggested that endothelial function is significantly improved in patients with leukocyte activation.

Effects of Lactotripeptide Supplementation on Tele-Monitored Home Blood Pressure and on Vascular and Renal Function in Prehypertension　- Randomized, Double-Blind, Placebo-Controlled, Cross-Over Study.

Background: This randomized, double-blind, placebo-controlled, cross-over study was conducted to examine the effects of lactotripeptide supplementation on 7-day mean tele-monitored home blood pressure (BP), and also on the markers of vascular function and renal damage in Japanese subjects with prehypertension. Methods and Results: A total of 26 subjects with prehypertension were randomly allocated to receive the active product (lactotripeptide tablet) or a placebo tablet for 8 weeks each in a cross-over manner. Urinary liver-type fatty acid-binding protein-to-creatine ratio (UFABPCR) and vascular function were measured at the end of each intervention. Home systolic and diastolic BP at the end of the lactotripeptide supplementation period was significantly lower than that at the end of the placebo period (P<0.05). On mixed linear model analysis there was a significant difference in the change in home diastolic BP after intervention between the 2 interventions (P=0.04). UFABPCR was significantly lower at the end of the lactotripeptide intervention period than at the end of the placebo period (P<0.05). Conclusions: The beneficial effect of lactotripeptide supplementation on 7-day mean tele-monitored home BP was confirmed in Japanese subjects with prehypertension. In addition, this intervention also seemed to have a protective effect against the progression of renal function decline.

Flow-mediated vasodilation as a diagnostic modality for vascular failure.

Vascular endothelial dysfunction represents an initial step of "vascular failure," which we have recently proposed as a comprehensive syndrome of failed vascular functions that extends from risk factors to established atherosclerotic disease. The early detection of vascular failure is essential in order to appropriately intervene and prevent its progression. Many efforts have been made to assess vascular endothelial function, and one of the most promising methods is the measurement of endothelium-dependent flow-mediated vasodilation (FMD) using high-frequency ultrasonographic imaging and transient occlusion of the brachial artery. The reactive hyperemia caused by the transient brachial arterial occlusion induces the release of local nitric oxide, resulting in vasodilation that can be quantified as an index of vasomotor function. The noninvasive nature of this technique allows repeated measurements over time to study the effectiveness of various interventions that may affect vascular health. Although there are technical and interpretive limitations of this technique, FMD-guided therapeutic approaches for vascular failure should contribute to the improvement of cardiovascular mortality and morbidity.

Administration of pigment epithelium-derived factor (PEDF) inhibits cold injury-induced brain edema in mice.

Brain edema is the most life-threatening complication that occurs as a result of a number of insults to the brain. However, its therapeutic options are insufficiently effective. We have recently found that administration of pigment epithelium-derived factor (PEDF) inhibits retinal hyperpermeability in rats by counteracting biological effects of vascular endothelial growth factor (VEGF). In this study, we investigated whether PEDF could inhibit cold injury-induced brain edema in mice. Cold injury was induced by applying a pre-cooled metal probe on the parietal skull. VEGF and its receptor Flk-1 gene and/or protein expressions were up-regulated in the cold-injured brain. Cold injury induced brain edema, which was reduced by intraperitoneal injection of VEGF antibodies (Abs) or apocynin, an inhibitor of NADPH oxidase. PEDF mRNA and protein levels were up-regulated in response to cold injury. PEDF dose-dependently inhibited the brain edema, whose effect was neutralized by simultaneous treatments with anti-PEDF Abs. Although VEGF and Flk-1 gene and/or protein expressions were not suppressed by PEDF, PEDF or anti-VEGF Abs inhibited the cold injury-induced NADPH oxidase activity in the brain. Further, PEDF treatment inhibited activation of Rac-1, an essential component of NADPH oxidase in the cold-injured brain, while it did not affect mRNA levels of gp91phox, p22phox, or Rac-1. These results demonstrate that PEDF could inhibit the cold injury-induced brain edema by blocking the VEGF signaling to hyperpermeability through the suppression of NADPH oxidase via inhibition of Rac-1 activation. Our present study suggests that PEDF may be a novel therapeutic agent for the treatment of brain edema.

Factors influencing trough and 90-minute plasma dabigatran etexilate concentrations among patients with non-valvular atrial fibrillation.

INTRODUCTION: Dabigatran etexilate, a direct oral anti-coagulation agent, is used in the prevention of thromboembolism in patients with non-valvular atrial fibrillation (NVAF). However, for reasons that are not fully understood, plasma dabigatran etexilate concentrations (PDC) vary significantly among patients. METHODS: We measured trough and 90min PDC in 98 patients with NVAF. To elucidate the cause of variations in PDC, we determined correlations between PDC and various factors including renal function, co-administration of a P-glycoprotein inhibitor, and the effects of three single nucleotide polymorphisms (SNPs) of the P-glycoprotein intestinal efflux transporter. To further determine the cause of PDC variations, we examined the relationship between PDC, activated partial prothrombin time (APTT), and D-dimer (DD) levels, which are surrogate markers for thrombotic risk. RESULTS: Multivariate analysis showed significant relations among creatinine, creatinine clearance, and CHA2D2-VaSc scores (p=0.04, p=0.01, and p=0.04, respectively). In addition, creatinine and creatinine clearance were significantly correlated with trough and 90min PDC (p<0.01), respectively. There was a clear linear relation between PDC and APTT, but not DD levels. However, higher DD levels (>0.5µg/mL) were associated with lower trough and 90min PDCs. CONCLUSIONS: Renal function and CHA2D2-VaSc scores affect PDC, suggesting these may be primary factors influencing the wide variation observed in PDCs under these conditions. Variations in APTT can primarily be explained by variations in PDC; patients with lower PDCs may have a higher risk of thromboembolism events.

Serum levels of advanced glycation end products (AGEs) are inversely associated with the number and migratory activity of circulating endothelial progenitor cells in apparently healthy subjects.

OBJECTIVES: Endothelial progenitor cells (EPCs) have been shown to participate in the process of vascular repair, thus playing a protective role against cardiovascular disease (CVD). It is known that atherosclerotic risk factors could affect EPC number and function. Advanced glycation end products (AGEs) contribute to the pathogenesis of atherosclerosis as well. However, as far as we know, there is no report to show the relationship between serum AGE levels and circulating EPCs in humans. Therefore, in this study, we investigated whether serum level of AGEs was associated with EPC number and functions in apparently healthy subjects, independent of traditional cardiovascular risk factors. RESEARCH DESIGN AND METHODS: Apparently healthy volunteers (34.6 ± 6.9 years old, 40 males and 8 females) who were not on any medications underwent a complete history and physical examination, determination of blood chemistries, including AGEs, and number, differentiation and migratory activity of circulating EPCs. RESULTS: Serum AGEs levels were 9.20 ± 1.85 U/mL. Multiple stepwise regression analysis revealed that serum levels of AGEs and smoking were independently correlated with reduced number of EPCs. Further, female, AGEs, and reduced HDL-cholesterol levels were independently associated with impaired migratory activity of circulating EPCs. CONCLUSIONS: This study demonstrated for the first time that the serum level of AGEs was one of the independent correlates of decreased cell number and impaired migratory activity of circulating EPCs in apparently healthy subjects. Our present observations suggest that even in young healthy subjects, serum level of AGEs may be a biomarker that could predict the progression of atherosclerosis and future cardiovascular events.

The p66shc gene expression in peripheral blood monocytes is increased in patients with coronary artery disease.

BACKGROUND: The p66(shc) protein has been shown to control cellular responses to oxidative stress, being involved in atherosclerosis in animal models. However, the relationship between the p66(shc) gene expression levels and coronary artery disease (CAD) in humans remains unknown. In this study, we examined whether the p66(shc) gene expression in peripheral blood monocytes (PBMs) was increased in patients with CAD, compared with age- and sex-matched subjects without CAD. HYPOTHESIS: We hypothesize that the p66(shc) gene expression level in PBMs is increased in patients with CAD. METHODS: Forty consecutive Japanese subjects who underwent coronary angiography for suspected CAD were enrolled in this study. The p66(shc) gene expression levels in PBMs were quantitatively measured by real-time reverse transcription-polymerase chain reactions. Uni- and multivariate analyses were applied for the correlates of CAD. CAD was diagnosed if there was > 75% obstruction of at least 1 major coronary artery or a history of percutaneous coronary intervention. RESULTS: There were no significant differences of blood chemistries and clinical characteristics between the patients with and without CAD, except the number of subjects who were on hypertension medication. The p66(shc) gene expression levels in PBMs were significantly higher in CAD patients compared with non-CAD subjects. Multiple stepwise regression analysis revealed that the p66(shc) gene expression levels and hypertension medication were independently related to CAD (R(2)=0.287). Further, the p66(shc) gene expres- sion levels were significantly increased (P < 0.05) in proportion to the number of diseased vessels. CONCLUSIONS: The present study is the first demonstration that increased the p66(shc) gene expression in PBMs is independently associated with CAD in Japanese subjects. The p66(shc) gene expression level in PBMs may be a novel biomarker of CAD in humans.

Factors associated with serum high mobility group box 1 (HMGB1) levels in a general population.

High mobility group box 1 (HMGB1), a nonhistone chromatin-associated protein, is implicated as a mediator of both infectious and non-infectious inflammatory conditions. Clinical research on this protein in humans just has begun; serum HMGB1 was reported to be elevated in a small number of critically ill patients suffering from sepsis. However, the kinetics, distribution and factors associated with circulating HMGB1 are unknown in a general population. In this study, we examined these issues in a large population of healthy subjects. Fasting blood samples were obtained from 626 subjects (237 males and 389 females). HMGB1 levels showed a skewed distribution with a mean of 1.65 +/- 0.04 ng/ml. Multiple stepwise regression analyses found that white blood cell (WBC) counts (P = .016) and the soluble form of receptor for advanced glycation end products (sRAGE; P < .001, inversely), which is also known to be a receptor for HMGB1, were independently associated with HMGB1 levels. We demonstrated for the first time that circulating HMGB1 levels were inversely associated with sRAGE levels in a general population. Because RAGE is involved in HMGB1 signaling, our present study suggests that sRAGE may capture and eliminate circulating HMGB1 in humans.

Low-density lipoprotein levels are one of the independent determinants of circulating levels of advanced glycation end products in nondiabetic subjects.

BACKGROUND: Nonenzymatic modification of proteins by reducing sugars leads to the formation of advanced glycation end products (AGEs), whose process has been reported to progress under diabetes. Recently, diet has been found to be a major environmental source of proinflammatory AGEs in humans. Further, fats or meat-derived products processed by high heat such as broiling have been shown to contain more AGEs than carbohydrates boiled for longer periods. Since circulating levels of low-density lipoprotein cholesterol (LDL-C) are also regulated by dietary cholesterol, it is conceivable that intake of cholesterol-rich foods could regulate serum levels of AGEs in humans. In this study, we investigated whether LDL-C levels are one of the independent determinants of circulating AGEs levels in a nondiabetic general population. METHODS: A total of 170 nondiabetic Japanese subjects underwent a complete history, physical examination, determination of blood chemistries, and serum AGEs. RESULTS: Univariate analysis showed that AGEs levels were associated with LDL-C (P < 0.05) and fasting plasma glucose levels (P < 0.05). By the use of multiple stepwise regression analyses, LDL-C (P < 0.01) and fasting plasma glucose levels (P < 0.05) remained significant and were independently related to AGEs levels (R2 = 0.069). CONCLUSIONS: The present study is the first demonstration that LDL-C levels are one of the independent determinants of serum levels of AGEs in a nondiabetic general population. Intake of cholesterol-rich foods may regulate serum levels of AGEs in nondiabetic subjects.

Pigment epithelium-derived factor (PEDF) administration inhibits occlusive thrombus formation in rats: a possible participation of reduced intraplatelet PEDF in thrombosis of acute coronary syndromes.

OBJECTIVES: Although remarkable therapeutic advances in the treatment of acute coronary syndromes (ACS) have been made with anti-platelet therapy, the therapeutic options may be limited by considerable side effects. Pigment epithelium-derived factor (PEDF) has anti-oxidative properties and may play a protective role against atherosclerosis. In this study, we investigated whether PEDF prevented occlusive thrombus formation in rats. METHODS AND RESULTS: Occlusive thrombus formation was induced by treating rats with ligation and cuff placement at the left common carotid artery. Intravenous injection of PEDF dose-dependently inhibited thrombus formation and blocked the increase in immunoreactivity of P-selectin, a marker of platelet activation, NADPH oxidase activity and superoxide generation in thrombi. In vitro, PEDF significantly decreased collagen-induced reactive oxygen species generation in platelets and subsequently suppressed the platelet activation and aggregation. Plasma and intraplatelet levels of PEDF in the coronary circulation in patients with ACS were significantly lower than those in age- and gender-matched controls without coronary artery disease. CONCLUSIONS: These results demonstrated that PEDF administration could inhibit occlusive thrombus formation by blocking the platelet activation and aggregation through its anti-oxidative properties. Our present study suggests that pharmacological up-regulation or substitution of PEDF may offer a promising strategy for the treatment of arterial thrombosis.

Daily exercise and bone marrow-derived CD34+/133+ cells after myocardial infarction treated by bare metal stent implantation.

BACKGROUND: The aims of the present study were to explore the mobilization of bone marrow-derived CD34(+)/133(+) cells in patients with acute myocardial infarction (AMI) and bare metal stent implantation who participated in daily exercise training, and associations with exercise capacity and restenosis. METHODS AND RESULTS: Participants comprised 23 Japanese men with AMI (Killip 1) who had been treated with a bare metal stent. All patients were advised to walk for 30-60 min/day, at least 4 times per week starting at 11 days after AMI, and were instructed to record the amount of time spent walking each day. At 10 days and then at 3 months after onset of AMI, symptom-limited cardiopulmonary exercise tests were performed and the number of CD34(+)/133(+) cells in the peripheral blood were measured by fluorescence-activated cell sorter analysis. At 3 months after AMI, the number of CD34(+)/133(+) cells and oxygen consumption at anaerobic threshold were higher in the high exercise group (ie, exercise duration >4 h/week) than the low exercise group (ie, exercise duration <2 h/week). At 3 months after AMI, the number of CD34(+)/133(+) cells significantly correlated with oxygen consumption at the anaerobic threshold (p=0.002). CONCLUSION: Moderate daily exercise of >4 h/week increases exercise capacity and the number of circulating CD34(+)/133(+) cells at 3 months after AMI.

Advanced glycation end products (AGEs) and cardiovascular disease (CVD) in diabetes.

Accelerated atherosclerosis and microvascular complications are perhaps the leading cause of coronary heart disease, blindness and renal failure, which could account for disabilities and high mortality rates in patients with diabetes. Several mechanisms including endothelial cell damage, platelet activation and aggregation, hypercoagulability, and impaired fibrinolysis are involved in the pathogenesis of thrombogenic diathesis in diabetes. However, the underlying molecular mechanism is not fully elucidated. A recent clinical study, the Diabetes Control and Complications Trial-Epidemiology of Diabetes Interventions and Complications (DCCT-EDIC) Research, has revealed that the reduction in the risk of progressive retinopathy and nephropathy resulting from intensive therapy in patients with type 1 diabetes persist for at least several years, despite increasing hyperglycemia. In addition, intensive therapy during the DCCT also reduced the risk of cardiovascular events by about 50 % in type 1 diabetic patients 11 years after the end of the trial. These clinical studies strongly suggest that so-called 'hyperglycemic memory' causes chronic abnormalities in diabetic vessels that are not easily reversed, even by subsequent, relatively good control of blood glucose. Among various biochemical pathways implicated in diabetic vascular complications, the process of formation and accumulation of advanced glycation end products (AGEs) and their mode of action are most compatible with the theory 'hyperglycemic memory'. In this review, we discuss the role of AGEs in thrombogenic abnormalities in diabetes, especially focusing on the deleterious effects of these macroproteins on endothelial cell function, platelet activation and aggregation, coagulation and fibrinolytic systems.

Pigment epithelium-derived factor inhibits neointimal hyperplasia after vascular injury by blocking NADPH oxidase-mediated reactive oxygen species generation.

Pigment epithelium-derived factor (PEDF) inhibits cytokine-induced endothelial cell activation through its antioxidative properties. However, the effect of PEDF on restenosis remains to be elucidated. Because the pathophysiological feature of restenosis is characterized by increased superoxide formation and accumulation of smooth muscle cells (SMCs), PEDF may inhibit this process via suppression of reactive oxygen species generation. We investigated here whether PEDF could prevent neointimal formation after balloon injury. PEDF levels were decreased in balloon-injured arteries. Adenoviral vector encoding human PEDF (Ad-PEDF) prevented neointimal formation. Expression and superoxide generation of the membrane components of NADPH oxidase, p22(phox) and gp91(phox), in the neointima were also suppressed by Ad-PEDF. Ad-PEDF reduced G(1) cyclin (cyclin D1 and E) expression and increased p27, a cyclin-dependent kinase inhibitor. In vitro, PEDF inhibited platelet-derived growth factor-BB-induced SMC proliferation and migration by blocking reactive oxygen species generation through suppression of NADPH oxidase activity via down-regulation of p22(PHOX) and gp91(PHOX). PEDF down-regulated G(1) cyclins and up-regulated p27 levels in platelet-derived growth factor-BB-exposed SMCs as well. These results demonstrate that PEDF could inhibit neointimal formation via suppression of NADPH oxidase-mediated reactive oxygen species generation. Our present study suggests that substitution of PEDF may be a novel therapeutic strategy for restenosis after balloon angioplasty.