Low-Dose Aspirin for Primary Prevention of Cardiovascular Events in Elderly Japanese Patients with Atherosclerotic Risk Factors: Subanalysis of a Randomized Clinical Trial (JPPP-70).

INTRODUCTION: This post hoc subanalysis of the randomized Japanese Primary Prevention Project investigated whether once-daily low-dose aspirin versus no aspirin reduced the risk of cardiovascular events (CVEs) in patients aged ≥ 70 years with atherosclerotic risk factors. METHODS: Patients aged < 70 years (young-old) or ≥ 70 years (old) with hypertension, dyslipidemia, or diabetes participated between 2005 and 2007. Patients were randomized 1:1 to receive 100 mg enteric-coated aspirin once daily or no aspirin plus standard of care. The primary outcome was a composite of death from cardiovascular causes plus nonfatal stroke and nonfatal myocardial infarction. The secondary outcome was a composite of the primary outcome plus transient ischemic attack, angina pectoris, and arteriosclerotic disease requiring medical or surgical intervention. Old (n = 7971) and young-old (n = 6493) patients were followed up for a median 5.02 years. RESULTS: Aspirin did not reduce the risk of primary (hazard ratio [HR] 0.92 [95% confidence interval {CI} 0.74-1.16]; P = 0.50) or secondary (0.85 [0.70-1.04]; P = 0.11) outcomes in patients aged ≥ 70 years. In old men with high-density lipoprotein < 40 mg/dL, treatment with low-dose aspirin was associated with a reduction in the incidence of the primary endpoint compared with the group not receiving aspirin (10/260 vs 22/250; HR 0.44 [95% CI 0.20-0.93]; P = 0.03). This subgroup was also found to contain significant larger proportions of patients with elevated body mass index, patients with diabetes mellitus, and smokers (P < 0.001). Old patients also showed differences in bleeding outcomes. Serious extracranial hemorrhage requiring transfusion or hospitalization occurred significantly more frequently in the aspirin-treated group than in the non-aspirin-treated group (35 [0.88%] vs 18 [0.45%]; HR 1.96 [1.11-3.46]; P = 0.020). Gastrointestinal hemorrhage occurred significantly more frequently in the aspirin-treated group than the non-aspirin-treated group (63 [1.58%] vs 18 [0.45%]; relative risk [RR] 3.5 [2.08-5.90]; P < 0.0001). Cerebral hemorrhage (intracranial hemorrhage) tended to occur more frequently in the aspirin-treated group than the non-aspirin-treated group (22 [0.55%] vs 11 [0.28%]; RR 2.01 [0.97-4.14]; P = 0.058). Cerebral hemorrhage occurred significantly more frequently in old patients than in young-old patients (33 [0.41%] vs 10 [0.15%]; HR 2.7 [1.34-5.53]; P = 0.0055). Gastrointestinal hemorrhage occurred in a slightly higher proportion of old patients compared with young-old patients (81 [1.02%] vs 53 [0.82%]; RR 1.2 [0.88-1.76]; P = 0.21). DISCUSSION/CONCLUSIONS: Aspirin did not reduce the risk of the primary or secondary outcomes in old patients. Aspirin treatment may have reduced CVEs within a high CVE risk elderly population subgroup. Aspirin treatment in such a group requires caution, because of the increased risk of intracranial hemorrhage, severe extracranial hemorrhage requiring hospitalization or transfusion, and gastrointestinal bleeding in old patients receiving aspirin therapy. CLINICAL TRIAL REGISTRATION: The study is registered at ClinicalTrials.gov [NCT00225849].

Photoswitchable Sn-Cyt c Solid-State Devices.

Electron transfer across proteins plays an important role in many biological processes, including those relevant for the conversion of solar photons to chemical energy. Previous studies demonstrated the generation of photocurrents upon light irradiation in a number of photoactive proteins, such as photosystem I or bacteriorhodopsin. Here, it is shown that Sn-cytochrome c layers act as reversible and efficient photoelectrochemical switches upon integration into large-area solid-state junctions. Photocurrents are observed both in the Soret band (λ = 405 nm) and in the Q band (λ = 535 nm), with current on/off ratios reaching values of up to 25. The underlying modulation in charge-transfer rate is attributed to a hole-transport channel created by the photoexcitation of the Sn-porphyrin.

Association of blood pressure control and metabolic syndrome with cardiovascular risk in elderly Japanese: JATOS study.

BACKGROUND: The impact of the metabolic syndrome (MS) on cardiovascular events in elderly subjects has not been clarified. We hypothesized that the impact differs between patients with and without strictly controlled blood pressure (BP) and also between early elderly (<75 years) and late (≥75 years) elderly patients. METHODS: Elderly hypertensive patients (65-85 years old) were randomly assigned to strict (target systolic BP <140 mm Hg) or mild (140-159 mm Hg) BP target, and were treated for 2 years with efonidipine-based regimen. MS was defined according to the National Cholesterol Education Program Adult Treatment Panel III criteria, except for the use of body mass index (BMI) ≥25 kg/m(2) instead of waist circumference. Primary endpoint was combined incidence of cardiovascular and renal events. Data were obtained from 2,865 patients. RESULTS: The prevalence of MS was 31.4%. The incidence of primary endpoint in patients with and without MS was 4.0% and 3.1%, respectively. MS was a significant risk factor for cardiovascular events in patients <75 years old (adjusted hazard ratio (HR) 2.17, P = 0.01), but not in patients ≥75 years old (adjusted HR 0.98, P = 0.94). In patients with MS, the event rate was significantly lower with strict treatment than with mild treatment among patients aged <75 years (P = 0.0006) but not in those aged ≥75 years (P = 0.82). CONCLUSIONS: MS was associated with cardiovascular risk in elderly hypertensive patients <75 years old, and strict BP control was beneficial for those with MS. However, MS and intensive control of BP may have little effect on cardiovascular events in elderly patients ≥75 years old.

Impact of renal function on cardiovascular events in elderly hypertensive patients treated with efonidipine.

This study evaluated the impact of renal function on cardiovascular outcomes in elderly hypertensive patients enrolled in the Japanese Trial to Assess Optimal Systolic Blood Pressure in Elderly Hypertensive patients. The patients were randomly assigned to either a strict-treatment group (target systolic blood pressure (BP) <140 mm Hg, n=2212) or a mild-treatment group (target systolic BP, 140 to <160 mm Hg, n=2206), each with efonidipine (a T/L-type Ca channel blocker)-based regimens. Cardiovascular events (stroke, cardiovascular disease and renal disease) were evaluated during the 2-year follow-up period following the prospective randomized open-blinded end-point method. Estimated glomerular filtration rate (eGFR) was elevated throughout the trial period in both the strict-treatment (59.4-62 ml min⁻¹ per 1.73 m²) and the mild-treatment group (58.8-61.4 ml min⁻¹ per 1.73 m²). This tendency was also observed in diabetic patients and patients aged ≥75 years, with baseline eGFR<60 ml min⁻¹ per 1.73 m². Baseline eGFR (<60 vs. ≥60 ml min⁻¹ per 1.73 m²) had no definite relationship with the incidence of cardiovascular events, nor did the level of BP control. Proteinuria at the time of entry into the study, however, was significantly correlated with cardiovascular event rates (7.1%), an association that was more apparent in patients with eGFR<60 ml min⁻¹ per 1.73 m² (8.2%). Furthermore, the event rate was more elevated in patients with greater declines in eGFR and was amplified when the baseline eGFR was <60 ml min⁻¹ per 1.73 m². In conclusion, the rates of decline of renal function and proteinuria constitute critical risk factors for cardiovascular events in elderly hypertensive patients, trends that are enhanced when baseline eGFR is diminished. Furthermore, the fact that efonidipine-based regimens ameliorate renal function in elderly hypertensive patients with chronic kidney disease may offer novel information on the mechanisms of cardiovascular protection.

A liposome-based energy conversion system for accelerating the multi-enzyme reactions.

We report the first example of a liposome-based energy conversion system that is useful for entrapping enzymes and NAD coenzyme to accelerate multi-step enzymatic reactions. The liposome generates a much higher catalytic current compared with the non-liposome system, which is in good consistency with numerical simulations.

A mediator-adapted diaphorase variant for a glucose dehydrogenase-diaphorase biocatalytic system.

Biofuel cell is an energy conversion device of the next generation which enables use of safer and higher energy-density fuels such as glucose. We have been developing a biofuel cell that comprises the three enzymes: glucose dehydrogenase (GDH) and diaphorase (DI) on anode, and bilirubin oxidase (BOD) on cathode. In this work, we have developed a DI variant suitable for our biofuel cell by using directed molecular evolution method. A gene library of DI variants was constructed by using error-prone PCR and the variant proteins were expressed in an Escherichia coli system. 8000 isolated variants have been screened with activity against 2-amino-1,4-naphthoquinone (ANQ), and 10 of them have been qualified which were then purified and examined their activities against ANQ. A highest activity was observed in G122D variant of which glycine residue at position 122 is substituted to aspartate. Enzymatic kinetic analyses show that KM for ANQ in G122D is 1/3 of that in wild type (G122D: 356 µM, wild type: 1.08 mM), whereas kcat and KM for NADH is almost the same, clearly showing that G122D mutation has given DI an improvement in enzymatic activity at lower ANQ concentration. The effect of this mutation was considered electrochemically in solution and in immobilized layer. The results show that G122D variant DI gave a higher current at lower ANQ concentration in solution, as well as in immobilized condition where GDH is co-immobilized within.

Comparison of strict- and mild-blood pressure control in elderly hypertensive patients: a per-protocol analysis of JATOS.

We performed a per-protocol analysis of the Japanese Trial to Assess Optimal Systolic Blood Pressure in Elderly Hypertensive Patients (JATOS) to evaluate the optimal target blood pressure (BP) in elderly hypertensive patients. In JATOS, conducted in elderly (65-85 years) hypertensive patients treated with efonidipine hydrochloride, there were no differences between the strict-treatment group (systolic BP maintained at <140 mm Hg) and the mild-treatment group (systolic BP maintained at ≥140 mm Hg and <160 mm Hg) in the incidence of primary end points (cardiovascular disease and renal failure) for 2 years. The present study analyzed data in subgroups of JATOS in which the average systolic BP was within the range of target values. The average BP levels achieved in the strict-target BP achieved subgroup (n=1191) and the mild-target BP achieved subgroup (n=1531) were 132.3/74.0 mm Hg and 146.6/78.3 mm Hg, respectively. The incidences of primary end points were similar between these subgroups (11.1/1000 patients per year and 13.2/1000 patients per year, respectively, P=0.502), and there were also no differences in the incidences of adverse events. The incidences of cardiovascular events in patients who failed to achieve their respective treatment goals, on the other hand, were significantly higher than in patients who achieved them. These results indicate that strict treatment for elderly hypertensive patients may have little effect in enhancing the suppression of the onset of cardiovascular events as compared with mild treatment, although patients who have difficulties in achieving treatment goals should be given more aggressive treatment as a high-risk population.

Impact of electrocardiographic left ventricular hypertrophy on the occurrence of cardiovascular events in elderly hypertensive patients. - The Japanese trial to assess optimal systolic blood pressure in elderly hypertensive patients (JATOS) -.

BACKGROUND: Electrocardiographic (ECG) left ventricular hypertrophy (LVH) is a risk factor for cardiovascular events and the incidence of LVH increases with age. However, few studies have assessed risks associated with LVH in elderly hypertensive patients. METHODS AND RESULTS: The Japanese Trial to Assess Optimal Systolic Blood Pressure in Elderly Hypertensive Patients (JATOS) was conducted to determine optimal blood pressure in elderly patients. At study entry, the sum of the S-wave in lead V(1) and the R-wave in lead V(5) (SV1+ RV5) could be determined in 3,230 patients, among whom 164 (5.1%) had cardiovascular events. On univariate analysis, the hazard ratio for cardiovascular events was 1.51 for each 10 mm (=1 mV) (95% confidence interval (CI): 1.34-1.69, P<0.0001) when SV1+ RV5 was considered a continuous variable, and 2.17 (95%CI: 1.54-3.05, P<0.0001) and 2.83 (95%CI: 1.91-4.19, P<0.0001) when SV1+ RV5 was classified into 2 groups at threshold values of either 35 mm or 40 mm, respectively. Multivariate Cox analysis showed that gender, age, current smoking, diabetes mellitus, history of renal disease, history of stroke, and SV1+ RV5 were significantly related to the occurrence of cardiovascular events. Kaplan - Meier curves showed that increasing SV1+ RV5 values were associated with higher incidences of cardiovascular events. CONCLUSIONS: ECG LVH is strongly related to cardiovascular events in elderly hypertensive patients.

Rationale, design, and baseline data of the Japanese Primary Prevention Project (JPPP)-a randomized, open-label, controlled trial of aspirin versus no aspirin in patients with multiple risk factors for vascular events.

BACKGROUND: Prevention of atherosclerotic disease has become an important public health priority in Japan due to the aging of the population and changes in diet and lifestyle factors. METHODS: The Japanese Primary Prevention Project (JPPP) is a multicenter, open-label, randomized, parallel-group trial that is evaluating primary prevention with low-dose aspirin in Japanese patients aged 60 to 85 years with hypertension, dyslipidemia, or diabetes mellitus. The study cohort will be followed for a mean of 4 years. The primary end point is a composite of death from cardiovascular causes (including fatal myocardial infarction [MI], fatal stroke, and other cardiovascular death), nonfatal stroke (ischemic or hemorrhagic), and nonfatal MI. Key secondary end points include a composite of cardiovascular death, nonfatal stroke, nonfatal MI, transient ischemic attack, angina pectoris, or arteriosclerotic disease requiring surgery or intervention; each component of the primary end point; noncerebrovascular and noncardiovascular death; and extracranial hemorrhage requiring transfusion or hospitalization. End point assessment is done by a central adjudication committee that is blinded to treatment assignments. RESULTS: Enrollment began in March 2005 and was completed in June 2007. A total of 14,466 patients were randomly allocated to receive enteric-coated aspirin, 100 mg/d, or no aspirin. At randomization, the study cohort had a mean (SD) age of 70.6 (6.2) years; 57.8% were women, 85.0% had hypertension, 71.7% had dyslipidemia, and 33.9% had diabetes. In the study cohort, 80.4% of patients had > or =3 risk factors. CONCLUSION: The JPPP is the largest primary prevention trial of aspirin in a Japanese population that is investigating whether the benefit of aspirin in reducing risk of vascular events outweighs any bleeding risk in elderly patients with multiple risk factors.

Mechanism of intramolecular electron transfer in the photoexcited Zn-substituted cytochrome c: theoretical and experimental perspective.

Photoinduced electron transfer (ET) in zinc-substituted cytochrome c (Zn-cyt c) has been utilized in many studies on the long-range ET in protein. Attempting to understand its ET mechanism in terms of electronic structure of the molecule, we have calculated an all-electron wave function for the ground-state of Zn-cyt c on the basis of density functional theory (DFT). The four molecular orbitals (MOs) responsible for excitation by UV-vis light (Gouterman's 4-orbitals) are assigned on the basis of the excited states of chromophore model for Zn-porphine complex calculated with the time-dependent DFT method. ET rates between each Gouterman's 4-orbitals and other MOs were estimated using Fermi's golden rule. It appeared that the two occupied MOs of the 4-orbitals show exclusively higher ET rate from/to particular MOs that localize on outermost amino acid residues (Lys 7 or Asn 54), respectively, whereas ET rates involving the two unoccupied MOs of the 4-orbitals are much slower. These results imply that the intramolecular ET in photoexcited Zn-cyt c is governed by the hole transfer through occupied MOs. The couplings of MOs between zinc porphyrin core and specific amino acid residues on the protein surface have been demonstrated in Zn-cyt c immobilized on an Au electrode via carboxylic acid group-terminated self-assembled monolayer. The Zn-cyt c-modified electrode showed photocurrents responsible for photoillumination. The action spectrum of the photocurrent was identical with the absorption spectrum of Zn-cyt c, indicating photoinduced electron conduction via occupied MOs. The voltage dependence of the photocurrent appeared to be linear and bidirectional like a photoconductor, which strongly supports the intramolecular ET mechanism in Zn-cyt c proposed on the basis of the theoretical calculations.

Primary prevention of cardiovascular disease with pravastatin in Japan (MEGA Study): a prospective randomised controlled trial.

BACKGROUND: Evidence-based treatment for hypercholesterolaemia in Japan has been hindered by the lack of direct evidence in this population. Our aim was to assess whether evidence for treatment with statins derived from western populations can be extrapolated to the Japanese population. METHODS: In this prospective, randomised, open-labelled, blinded study, patients with hypercholesterolaemia (total cholesterol 5.69-6.98 mmol/L) and no history of coronary heart disease or stroke were randomly assigned diet or diet plus 10-20 mg pravastatin daily. The primary endpoint was the first occurrence of coronary heart disease. Statistical analyses were done by intention to treat. This trial is registered at ClinicalTrials.gov, number NCT00211705. FINDINGS: 3966 patients were randomly assigned to the diet group and 3866 to the diet plus pravastatin group. Mean follow-up was 5.3 years. At the end of study, 471 and 522 patients had withdrawn, died, or been lost to follow-up in the diet and diet plus pravastatin groups, respectively. Mean total cholesterol was reduced by 2.1% (from 6.27 mmol/L to 6.13 mmol/L) and 11.5% (from 6.27 mmol/L to 5.55 mmol/L) and mean LDL cholesterol by 3.2% (from 4.05 mmol/L to 3.90 mmol/L) and 18.0% (from 4.05 mmol/L to 3.31 mmol/L) in the diet and the diet plus pravastatin groups, respectively. Coronary heart disease was significantly lower in the diet plus pravastatin group than in the diet alone group (66 events vs 101 events; HR 0.67, 95% CI 0.49-0.91; p=0.01). There was no difference in the incidence of malignant neoplasms or other serious adverse events between the two groups. INTERPRETATION: Treatment with a low dose of pravastatin reduces the risk of coronary heart disease in Japan by much the same amount as higher doses have shown in Europe and the USA.

Binding of dioxygen to non-metal sites in proteins: exploration of the importance of binding site size versus hydrophobicity in the copper amine oxidase from Hansenula polymorpha.

Copper amine oxidases (CAOs) contain 2,4,5-trihydroxyphenylalanyl quinone (TPQ) and a copper ion in their active sites, catalyzing amine oxidation to aldehyde and ammonia concomitant with the reduction of molecular oxygen to hydrogen peroxide. Kinetic studies on the CAO from bovine serum (BSAO) [Su and Klinman (1999) Biochemistry 37, 12513-12525] and the recent reports on the cobalt substituted form of the enzyme from Hansenula polymorpha (HPAO) [Mills and Klinman (2000) J. Am. Chem. Soc. 122, 9897-9904, and Mills et al. (2002) Biochemistry, 41, 10577-10584] support pre-binding of molecular oxygen prior to a rate-limiting electron transfer from the reduced form of TPQ (p-aminohydroquinone form) to dioxygen. Although there is significant sequence homology between BSAO and HPAO, k(cat)/K(m)(O2) for BSAO under the optimal condition is one order of magnitude lower than that for HPAO. From a comparison of amino acid sequences for BSAO and HPAO, together with the X-ray crystal structure of HPAO, a plausible dioxygen pre-binding site has been identified that involves Y407, L425, and M634 in HPAO; the latter two residues are altered in BSAO to A490 and T695. To determine which of these residues plays a greater role in dioxygen chemistry, k(cat)/K(m)(O2) was determined in HPAO for the M634 --> T and L425 --> A mutants. The L425 --> A mutation does not alter k(cat)/K(m)(O2) to a large extent, whereas the M634 --> T decreased k(cat)/K(m)(O2) by one order of a magnitude, creating a catalyst that is similar to BSAO. A series of mutants at M634 (to F, L, and Q) were, therefore, prepared in HPAO and characterized with regard to k(cat)/K(m)(O2) as a function of pH. Structure reactivity correlations show a linear relationship of rate with side chain volume, rather than hydrophobicity, indicating that dioxygen reactivity increases with the bulk of the residue at position 634. This site also shows specificity for O2, in relation to the co-gas N2, since substitution of the inert gas N2 by either Ar or He has no effect on measured rates. In particular, He gas is expected to have little affinity for protein at 1 atmospheric pressure, implying little or no binding by N2 as well.

Mechanistic comparison of the cobalt-substituted and wild-type copper amine oxidase from Hansenula polymorpha.

A recent report by Mills and Klinman [Mills, S. A., and Klinman, J. P. (2000) J. Am. Chem. Soc. 122, 9897-9904] described the preparation and initial characterization of a cobalt-substituted form of the copper amine oxidase from Hansenula polymorpha (HPAO). This enzyme was found to be fully catalytically active at saturating substrate concentrations, but with a K(m) for O(2) approximately 70-fold higher than that of the copper-containing, wild-type enzyme. Herein, we report a detailed analysis of the mechanism of catalysis for the wild-type and the cobalt-substituted forms of HPAO. Both forms of enzyme are concluded to utilize the same mechanism for oxygen reduction, involving initial, rate-limiting electron transfer from the reduced cofactor of the enzyme to prebound dioxygen. Superoxide formed in this manner is stabilized by the active site metal, facilitating the transfer of a second electron and two protons to form the product hydrogen peroxide. The elevated K(m) for O(2) at the dioxygen binding site in Co-substituted HPAO, relative to that of wild-type HPAO, is proposed to be due to a change in the net charge at the adjacent metal site from +1 (cupric hydroxide) in wild-type enzyme to +2 (cobaltous H(2)O) in cobalt-substituted HPAO.