Hisashi Makino

Relationship Between Blood Pressure Category and Incidence of Stroke and Myocardial Infarction in an Urban Japanese Population With and Without Chronic Kidney Disease: The Suita Study

Background and Purpose— Chronic kidney disease (CKD) is increasingly recognized as an independent risk factor for stroke and myocardial infarction (MI). Few studies, however, have examined the relationship between blood pressure (BP) category and these diseases in subjects with and without CKD. Methods— We studied 5494 Japanese individuals (ages 30 to 79, without stroke or MI at baseline) who completed a baseline survey and received follow-up through December 2005. The glomerular filtration rate (GFR) was estimated using the Modification of Diet in Renal Disease study equation modified by the Japanese coefficient. CKD was defined as an estimated GFR <60 mL/min/1.73m2. BP categories were defined by the European Society of Hypertension and European Society of Cardiology 2007 criteria. Results— In 64 395 person-years of follow-up, we documented 346 incidences of cardiovascular diseases (CVD; 213 strokes and 133 MI events). Compared with the GFR (≥90 mL/min/1.73m2) group, the hazard ratios (95% confidential intervals) for stroke were 1.9 (1.3 to 3.0) in the GFR 50 to 59 mL/min/1.73m2 group and 2.2 (1.2 to 4.1) in the GFR <50 mL/min/1.73m2 group. Results for cerebral infarction were similar. Compared with the optimal BP subjects without CKD, the normal BP, high-normal BP, and hypertensive subjects without CKD showed increased risks of CVD and stroke; however the impact of each BP category on CVD (P for interaction: 0.04 in men, 0.49 in women) and stroke (0.03 in men, 0.90 in women) was more evident in men with CKD. Conclusions— CKD may increase the association of BP and CVD in a Japanese urban population.

Altered gene expression related to glomerulogenesis and podocyte structure in early diabetic nephropathy of db/db mice and its restoration by pioglitazone

Glomerular injury plays a pivotal role in the development of diabetic nephropathy. To elucidate molecular mechanisms underlying diabetic glomerulopathy, we compared glomerular gene expression profiles of db/db mice with those of db/m control mice at a normoalbuminuric stage characterized by hyperglycemia and at an early stage of diabetic nephropathy with elevated albuminuria, using cDNA microarray. In db/db mice at the normoalbuminuric stage, hypoxia-inducible factor-1α (HIF-1α), ephrin B2, glomerular epithelial protein 1, and Pod-1, which play key roles in glomerulogenesis, were already upregulated in parallel with an alteration of genes related to glucose metabolism, lipid metabolism, and oxidative stress. Podocyte structure-related genes, actinin 4α and dystroglycan 1 (DG1), were also significantly upregulated at an early stage. The alteration in the expression of these genes was confirmed by quantitative RT-PCR. Through pioglitazone treatment, gene expression of ephrin B2, Pod-1, actinin 4α, and DG1, as well as that of oxidative stress and lipid metabolism, was restored concomitant with attenuation of albuminuria. In addition, HIF-1α protein expression was partially attenuated by pioglitazone. These results suggest that not only metabolic alteration and oxidative stress, but also the alteration of gene expression related to glomerulogenesis and podocyte structure, may be involved in the pathogenesis of early diabetic glomerulopathy in type 2 diabetes.

Long-term Effect of Low-density Lipoprotein Apheresis in Patients with Homozygous Familial Hypercholesterolemia

Abstract:  Patients that are homozygous for familial hypercholesterolemia (FH) exhibit severe hypercholesterolemia, cutaneous and tendon xanthomas and premature atherosclerosis beginning in childhood. They are resistant to drug therapy and low‐density lipoprotein (LDL) apheresis is the practical treatment. Here we review the technique of LDL apheresis treatment, the long‐term effects of LDL apheresis, the effect of apheresis on pregnancy, and the drugs that have proven beneficial in patients with homozygous FH. We also record our experiences of treating eight homozygous FH patients using the LDL apheresis treatment. Among the eight patients, one has been free from cardiovascular disease and two patients have each regressed once. In two patients, aortic valve stenosis developed and the other two patients died for acute myocardial infarction. Furthermore, two patients delivered healthy babies in spite of coronary artery disease. Thus, LDL apheresis therapy has the possibility of preventing the progression of atherosclerosis, but the prognosis assessed by long‐term observation is still not satisfactory. A recent clinical trial showed some efficacy of the combination therapy of LDL apheresis and atorvastatin for reducing serum choresterol levels in homozygous FH, suggesting that this combination therapy may be useful for prevention of atherosclerosis in patients homozygous for FH.

Characterization of Autosomal Dominant Hypercholesterolemia Caused by PCSK9 Gain of Function Mutations and its Specific Treatment with Alirocumab, a PCSK9 Monoclonal Antibody

Background—Patients with PCSK9 gene gain of function (GOF) mutations have a rare form of autosomal dominant hypercholesterolemia. However, data examining their clinical characteristics and geographic distribution are lacking. Furthermore, no randomized treatment study in this population has been reported. Methods and Results—We compiled clinical characteristics of PCSK9 GOF mutation carriers in a multinational retrospective, cross-sectional, observational study. We then performed a randomized placebo-phase, double-blind study of alirocumab 150 mg administered subcutaneously every 2 weeks to 13 patients representing 4 different PCSK9 GOF mutations with low-density lipoprotein cholesterol (LDL-C) ≥70 mg/dL on their current lipid-lowering therapies at baseline. Observational study: among 164 patients, 16 different PCSK9 GOF mutations distributed throughout the gene were associated with varying severity of untreated LDL-C levels. Coronary artery disease was common (33%; average age of onset, 49.4 years), and untreated LDL-C concentrations were higher compared with matched carriers of mutations in the LDLR (n=2126) or apolipoprotein B (n=470) genes. Intervention study: in PCSK9 GOF mutation patients randomly assigned to receive alirocumab, mean percent reduction in LDL-C at 2 weeks was 62.5% (P<0.0001) from baseline, 53.7% compared with placebo-treated PCSK9 GOF mutation patients (P=0.0009; primary end point). After all subjects received 8 weeks of alirocumab treatment, LDL-C was reduced by 73% from baseline (P<0.0001). Conclusions—PCSK9 GOF mutation carriers have elevated LDL-C levels and are at high risk of premature cardiovascular disease. Alirocumab, a PCSK9 antibody, markedly lowers LDL-C levels and seems to be well tolerated in these patients. Clinical Trial Registration—URL: http://www.clinicaltrials.gov. Unique Identifier: NCT01604824.

Decreased circulating CD34+ cells are associated with progression of diabetic nephropathy

Aims  Circulating progenitor cells such as CD34+ cells play a key role in maintenance of vascular endothelial function and neovascularization, and a decrease in the number of CD34+ cells is associated with cardiovascular disease. However, the contribution of circulating progenitor cells to microvascular disease, such as diabetic nephropathy, is unclear. This study was therefore designed to clarify the association between diabetic nephropathy and circulating CD34+ cells.

Removal of Plasma Mature and Furin-Cleaved Proprotein Convertase Subtilisin/Kexin 9 by Low-Density Lipoprotein-Apheresis in Familial Hypercholesterolemia: Development and Application of a New Assay for PCSK9

CONTEXT Proprotein convertase subtilisin/kexin 9 (PCSK9) is known to be a good target to decrease LDL cholesterol (LDL-C) and two forms of PCSK9, mature and furin-cleaved PCSK9, circulate in blood. However, it has not been clarified whether and how the levels of each PCSK9 are affected by LDL-apheresis (LDL-A) treatment, a standard therapy in patients with severe forms of familial hypercholesterolemia (FH). OBJECTIVE Our objective was to investigate the differences in LDL-A-induced reduction of mature and furin-cleaved PCSK9 between homozygous and heterozygous FH, and between dextran sulfate (DS) cellulose adsorption and double membrane (DM) columns and to clarify the mechanism of their removal. DESIGN A sandwich ELISA to measure two forms of PCSK9s using monoclonal antibodies was developed. Using the ELISA, PCSK9 levels were quantified before and after LDL-A with DS columns in 7 homozygous and 11 heterozygous FH patients. A crossover study between the two column types was performed. The profiles of PCSK9s were analyzed after fractionation by gel filtration chromatography. Immunoprecipitation of apolipoprotein B (apoB) in FH plasma was performed. RESULTS Both mature and furin-cleaved PCSK9s were significantly decreased by 55-56% in FH homozygotes after a single LDL-A treatment with DS columns, and by 46-48% or 48-56% in FH heterozygotes after treatment with DS or DM columns. The reduction ratios of LDL-C were strongly correlated with that of PCSK9 in both FH homozygotes and heterozygotes. In addition, more than 80% of plasma PCSK9s were in the apoB-deficient fraction and a significant portion of mature PCSK9 was bound to apoB, as shown by immunoprecipitation. CONCLUSIONS Both mature and furin-cleaved PCSK9s were removed by LDL-A in homozygous and heterozygous FH either by binding to apoB or by other mechanisms. The ELISA method to measure both forms of plasma PCSK9 would be useful for investigating physiological or pathological roles of PCSK9.

Pioglitazone treatment stimulates circulating CD34-positive cells in type 2 diabetes patients

Circulating bone marrow derived immature cells, including CD34-positive (CD34(+)) cells, contribute to maintenance of the vasculature, not only as a pool of endothelial progenitor cells (EPCs), but also as a source of growth/angiogenesis factor. We hypothesized that the thiazolidineone compound pioglitazone could stimulate the circulating CD34(+) cells in diabetic patients. Thirty-four patients with type 2 diabetes received 15-30 mg pioglitazone for 24 weeks. The number of circulating CD34(+) cells significantly increased at 12 and continued this effect for 24 weeks (1.08+/-0.39, 1.34+/-0.34 and 1.32+/-0.28cells/microl at 0, 12 and 24 weeks, respectively). The change of CD34(+) cell levels (DeltaCD34(+) cells) between 0 and 12 weeks was significantly correlated with the change of high sensitive C reactive protein levels (Deltahs-CRP) and change in adiponectin levels (Deltaadiponectin) (r=-0.412, r=0.359, respectively). Our study demonstrated that pioglitazone treatment increased circulating CD34(+) cells, suggesting that this effect may at least partly contribute to the anti-atherosclerotic action of pioglitazone.

Proteomic Analysis of Proteins Eliminated by Low‐Density Lipoprotein Apheresis

Low‐density lipoprotein apheresis (LDL‐A) treatment has been shown to decrease serum LDL cholesterol levels and prevent cardiovascular events in homozygous patients with familial hypercholesterolemia. Recently, LDL‐A treatment has been suggested to have beneficial effects beyond the removal of LDL particles. In this study, to clarify the preventive effects of LDL‐A treatment on atherosclerosis, the waste fluid from the adsorption columns was analyzed. The waste fluid of LDL adsorption columns was analyzed by two‐dimensional electrophoresis followed by mass spectrometry. Serum concentrations of the newly identified proteins before and after LDL‐A treatment were measured by enzyme‐linked immunosorbent assay. We identified 48 kinds of proteins in the waste fluid of LDL adsorption columns, including coagulation factors, thrombogenic factors, complement factors, inflammatory factors and adhesion molecules. In addition to the proteins that were reported to be removed by LDL‐A treatment, we newly identified several proteins that have some significant roles in the development of atherosclerosis, including vitronectin and apolipoprotein C‐III (Apo C‐III). The serum levels of vitronectin and Apo C‐III decreased by 82.4% and 54.8%, respectively, after a single LDL‐A treatment. While Apo C‐III was removed with very low‐density lipoprotein (VLDL) and LDL, vitronectin was removed without association with lipoproteins. The removal of proteins observed in the waste fluid has a certain impact on their serum levels, and this may be related to the efficacy of LDL‐A treatment. Proteomic analysis of the waste fluid of LDL adsorption columns may provide a rational means of assessing the effects of LDL‐A treatment.

Visceral fat is negatively associated with B-type natriuretic peptide levels in patients with advanced type 2 diabetes

AIMS The association between BMI and low levels of B-type natriuretic peptide (BNP), a marker of heart failure, has been demonstrated in a large population-based cohort. We examined the effects of obesity on BNP levels in patients with diabetes that are often associated with obesity and a higher risk for heart failure. METHODS Plasma BNP levels, BMI, and cardiac function parameters were measured in 608 patients with type 2 diabetes. A computed tomography scan was performed to measure abdominal fat. RESULTS In multivariable regression analyses adjusted for age, sex, systolic blood pressure, pulse rate, serum creatinine, asynergy, left atrial dimension, percent fractional shortening, and left ventricular mass, there was an inverse relationship between BMI and BNP (p<0.001). Obese individuals with 25</=BMI<30 and 30</=BMI individuals were more likely to have lower BNP levels compared with BMI<22 individuals (multivariable-adjusted odds ratios (95% CIs): 1.61 (1.16-2.26) and 2.07 (1.35-3.22), respectively). Inverse associations were noted between BNP and visceral fat area (VFA) in both sexes (p=0.029 for men, p=0.024 for women). CONCLUSIONS In patients with type 2 diabetes, BNP levels are significantly lower in obese subjects after multivariable adjustments. Among various obesity parameters, visceral fat was most closely associated with BNP levels.

Decreased levels of circulating CD34+ cells are associated with coronary heart disease in Japanese patients with type 2 diabetes

Circulating progenitor cells, including CD34 positive (CD34+) cells, play a key role in neovascularisation and the maintenance of vascular endothelial function. Several lines of evidence show an association between decreased levels of circulating CD34+ cells and cardiovascular disease. However, the contribution of circulating CD34+ cells to the occurrence of cardiovascular events in diabetic patients remains unclear.