Yoshihiro Maekawa

Klotho suppresses TNF-α-induced expression of adhesion molecules in the endothelium and attenuates NF-κB activation

Klotho is a senescence suppressor protein that, when overexpressed, extends the lifespan of mice. Klotho-disrupted mice exhibit atherosclerosis and endothelial dysfunction, which led us to investigate the effect of the Klotho protein on vascular inflammation, particularly adhesion molecule expression. In this study, human umbilical vein endothelial cells (HUVECs) were preincubated with Klotho protein and then exposed to tumor necrosis factor-α (TNF-α) or vehicle. Reverse transcription-PCR and Western blot analyses revealed that Klotho suppressed TNF-α-induced expression of intracellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). NF-κB activation, IκB phosphorylation induced by TNF-α were also attenuated by Klotho protein administration. The inhibition of eNOS phosphorylation by TNF-α was reversed by Klotho. Furthermore, Klotho inhibited TNF-α-induced monocyte adhesion to HUVECs and suppressed adhesion molecule expression in an organ culture of the rat aorta. These results suggest that Klotho suppresses TNF-α-induced expression of adhesion molecules and NF-κB activation. Klotho may have a role in the modulation of endothelial inflammation.

Klotho protein diminishes endothelial apoptosis and senescence via a mitogen-activated kinase pathway.

Aim:  Mice that carry the Klotho mutation (KL‐/‐) manifest diverse age‐related disorders similar to those observed in humans. Thus, the Klotho protein might function as an anti‐aging hormone in mammals. Recently, we reported that Klotho recombinant protein attenuated apoptosis and cellular senescence in endothelial cells, but the mechanism remained unclear. Here, we designed an in vitro study to test whether inhibitors of extracellular signal‐regulated kinase and mitogen‐activated kinase kinase could affect Klotho regulation of apoptosis and cellular senescence.

Loss of ACE2 accelerates time-dependent glomerular and tubulointerstitial damage in streptozotocin-induced diabetic mice.

As angiotensin-converting enzyme-2 (ACE2) was identified as a negative regulator of the renin–angiotensin system, there have been many reports concerning its role in several tissues, including the kidney. However, the role of ACE2 during the development of diabetic nephropathy remains undetermined, as previous reports did not necessarily support a protective role against renal injury. Thus, we performed detailed observations of kidneys in ACE2-knockout (ACE2-KO) mice at early (4 weeks) and advanced (18 weeks) stages of diabetes. ACE2-KO and wild-type C57BL/6 mice were rendered diabetic by intraperitoneal injection of streptozotocin. Diabetic ACE2-KO mice showed earlier onset and more severe progression of albuminuria than those did wild-type mice. The elevation of serum creatinine and urea nitrogen levels at 18 weeks of diabetes was more prominent in ACE2-KO mice. Periodic acid-Schiff-stained cross-section of diabetic ACE2-KO mice showed a more severe time-dependent increase in glomerular/tubulointerstitial damage than did that of wild-type mice, confirmed by the immunostaining of α-smooth muscle actin, collagen IV and F4-80 antigen. Glomeruli of diabetic ACE2-KO mice showed earlier and more severe decrease in the expression of nephrin, whose degradation is involved in the onset of albuminuria, and more potent increase of vascular endothelial growth factor expression. In addition, treatment with AT1 receptor blocker olmesartan significantly, but not totally, ameliorated the functional and morphological deterioration of diabetic nephropathy in ACE2-KO mice. These results suggest that ACE2 might continuously protect from both glomerular and tubulointerstitial injury during the development of diabetic nephropathy. The renal-protective effect of ACE2 might involve more than just suppressing angiotensin II-mediated AT1 receptor signaling.

Effects of hydroxyhydroquinone-reduced coffee on vasoreactivity and blood pressure

Recent studies suggest that chlorogenic acids, which are the main components of the polyphenol class in coffee, decrease blood pressure, and that hydroxyhydroquinone (HHQ), which is generated by roasting coffee beans, inhibits the antihypertensive effect of chlorogenic acids in brewed coffee. Here, we examined the vasoreactivity and antihypertensive effects of HHQ-reduced coffee in mild hypertension. The study design was a double blind, randomized, placebo-controlled intervention study, with a 4-week run-in period, followed by an 8-week test beverage ingestion period. The subjects were Japanese men and women with mild hypertension and vascular failure, who were not taking any antihypertensive drugs. During the test beverage ingestion period, the subjects ingested either active or placebo HHQ-reduced coffee (chlorogenic acids per 184 ml of coffee: active, 300 mg; and placebo, 0 mg) daily. Subjects were randomly divided into two groups: active group (n=9) and placebo group (n=12). In the active beverage group, endothelium-dependent, flow-mediated vasodilation impairment was significantly ameliorated and systolic blood pressure was significantly decreased from the baseline, but not in the placebo group. There were no test beverage consumption-related changes in other parameters that may influence blood pressure, such as pulse, cardiac output, body weight or 24-h urine volume. Ingestion of the active beverage significantly decreased urinary isoprostane levels, suggesting a reduced oxidative stress. These findings indicate that HHQ-reduced coffee decreased blood pressure in subjects with mild hypertension. The decreased blood pressure was associated with improved vascular endothelial function.

β -Adrenergic receptor gene polymorphism is a genetic risk factor for cardiovascular disease: a cohort study with hypertensive patients

Single-nucleotide polymorphisms (SNPs) of the β-adrenergic receptor (βADR) subtypes are related to hypertension and obesity. This hospital-based cohort study with hypertensive patients evaluated five βADR SNPs in association with cardiovascular events. The cohort included 357 hypertensive patients (male=181; mean age=61.5±11.8 years) seen between January 1998 and June 2004. The SNPs (Ser49Gly and Arg389Gly for β1ADR; Gly16Arg and Glu27Gln for β2ADR; Trp64Arg for β3ADR) were identified by PCR. We used Kaplan–Meier curves to assess the prognostic effect of these SNPs on cardiovascular disease (CVD). The SNP frequencies were Ser/Ser:Ser/Gly:Gly/Gly=243:104:10; Arg/Arg:Arg/Gly:Gly/Gly=256:95:6; Gly/Gly:Gly/Arg:Arg/Arg=71:201:85; Gln/Gln:Glu/Gln=308:49; and Trp/Trp:Trp/Arg:Arg/Arg=265:89:3. A total of 17 stroke and 15 coronary artery disease cases were recorded. By Kaplan–Meier analysis, the Ser/Ser SNP in Ser49Gly (P=0.0398), the Glu/Gln SNP in Glu27Gln (P=0.0390) and the Trp/Trp SNP in Trp64Arg (P=0.0132) were associated with lower event-free CVD survival (log-rank, Mantel–Cox model). A Cox proportional hazards model revealed that only the Trp/Trp SNP (P=0.0321) and age (P=0.0186) were independently related to lower event-free survival for CVD, adjusted for gender, diabetes, dyslipidemia, blood pressure, body mass index, medication and hypertensive complications. Combination Kaplan–Meier analysis of these three positive SNPs indicated a higher frequency of CVD among patients with the combination of Ser/Ser in Ser49Gly of β1, Glu/Gln in Glu27Gln of β2 and Trp/Trp in Trp64Arg of β3 (P=0.0209). These three SNPs, especially the Trp64Arg SNP of β3ADR, might be risk factors for CVD in hypertensive patients.

The combination of chronic kidney disease and increased arterial stiffness is a predictor for stroke and cardiovascular disease in hypertensive patients

To clarify the clinical utility of pulse wave velocity (PWV) and chronic kidney disease (CKD) in hypertension, we analyzed the prognostic impact of PWV and CKD on cerebrocardiovascular disease in hypertensive patients. This study consisted of 531 patients with essential hypertension (male/female=292/239, mean age=61.7±12.3, mean follow-up=7.0±3.0 years) and was performed between January 1998 and June 2004. We used questionnaires to assess stroke (n=57), cardiovascular diseases (CVDs; myocardial infarction, angina and congestive heart failure; n=44) and death (n=53) as primary end points. At baseline, we evaluated the carotid–femoral PWV (9.1±1.8 m s−1), the glomerular filtration rate and urinary protein excretions. We divided these subjects into those in the highest quartile of PWV and other subjects and into CKD (n=149) and non-CKD (n=458). We evaluated the prognostic influences of PWV and CKD with Kaplan–Meier analysis and Cox’s proportional hazard model. PWV in CKD (9.6±1.9 m s−1) was higher than in non-CKD (8.8±1.6 m s−1; P<0.0001), and creatinine was slightly decreased in the highest PWV group (1.09±0.35 mg dl−1, P<0.0001). On the basis of Kaplan–Meier analysis, the highest PWV group (PWV>10.1 m s−1; P=0.0003) and the CKD group (P=0.0005) showed significantly higher proportions of stroke and CVD events. In addition, the highest PWV group showed the highest percentage of stroke (P=0.0007), and the CKD group showed the highest proportion of CVD (P<00001). High PWV and CKD were independent predictors for stroke and CVD (P=0.0332) by Cox’s proportional hazard model. These data suggest that increased aortic stiffness and CKD may be predictors for stroke and cardiovascular events in hypertensive patients.

Oxidized LDL (oxLDL) activates the angiotensin II type 1 receptor by binding to the lectin-like oxLDL receptor

The angiotensin II type 1 receptor (AT1) is a 7‐transmembrane domain GPCR that when activated by its ligand angiotensin II, generates signaling events promoting vascular dysfunction and the development of cardiovascular disease. Here, we show that the single‐transmembrane oxidized LDL (oxLDL) receptor (LOX‐1) resides in proximity to AT1 on cell‐surface membranes and that binding of oxLDL to LOX‐1 can allosterically activate AT1‐dependent signaling events. oxLDL‐induced signaling events in human vascular endothelial cells were abolished by knockdown of AT1 and inhibited by AT1 blockade (ARB). oxLDL increased cytosolic G protein by 350% in Chinese hamster ovary (CHO) cells with genetically induced expression of AT1 and LOX‐1, whereas little increase was observed in CHO cells expressing only LOX‐1. Immunoprecipitation and in situ proximity ligation assay (PLA) assays in CHO cells revealed the presence of cell‐surface complexes involving LOX‐1 and AT1. Chimeric analysis showed that oxLDLinduced AT1 signaling events are mediated via interactions between the intracellular domain of LOX‐1 and AT1 that activate AT1. oxLDL induced impairment of endothelium‐dependent vascular relaxation of vascular ring from mouse thoracic aorta was abolished by ARB or genetic deletion of AT1. These findings reveal a novel pathway for AT1 activation and suggest a new mechanism whereby oxLDL may be promoting risk for cardiovascular disease.—Yamamoto, K., Kakino, A., Takeshita, H., Hayashi, N., Li, L., Nakano, A., Hanasaki‐Yamamoto, H., Fujita, Y., Imaizumi, Y., Toyama‐Yokoyama, S., Nakama, C., Kawai, T., Takeda, M., Hongyo, K., Oguro, R., Maekawa, Y., Itoh, N., Takami, Y., Onishi, M., Takeya, Y., Sugimoto, K., Kamide, K., Nakagami, H., Ohishi, M., Kurtz, T. W., Sawamura, T., Rakugi, H. Oxidized LDL (oxLDL) activates the angiotensin II type 1 receptor by binding to the lectin‐like oxLDL receptor. FASEB J. 29, 3342‐3356 (2015). www.fasebj.org

Angiotensin-converting enzyme single nucleotide polymorphism is a genetic risk factor for cardiovascular disease : a cohort study of hypertensive patients

The renin-angiotensin system (RAS) adversely affects stroke and cardiovascular disease; polymorphisms in genes involved in this system are associated with cardiovascular disease. The aim of the present study was to confirm the genetic risk of these polymorphisms for stroke and cardiovascular events in a cohort study of 515 hypertensive patients in Japan (follow-up period 90.6±30.2 months). The insertion/deletion (I/D) polymorphism of the gene encoding angiotensin-converting enzyme (ACE), the M235T amino acid change in angiotensinogen, and the A1166C polymorphism in angiotensin II type 1 receptor were determined by TaqMan PCR. In Kaplan–Meier analyses, the ACE I/D polymorphism was a risk factor for cerebro-cardiovascular events, especially cardiovascular events (P<0.0001), and the M235T mutation was a risk factor for cardiovascular events (P<0.0105). The cumulative rates of cerebro-cardiovascular end points for the ACE polymorphism were 10.6, 16.4 and 42.2% for the II (n=207), ID (n=244) and DD (n=64) genotype carriers, respectively (P<0.0001). Cox’s proportional hazard models revealed that the ACE DD genotype was a risk factor for cerebro-cardiovascular and cardiovascular events (after adjusting for common risk factors), anti-hypertensive treatment and RAS inhibition (P<0.0001). Moreover, after adjustment for the common risk factors left ventricular hypertrophy and previous myocardial infarction/stroke, these phenomena were preserved. Thus, the DD genotype of ACE may be a genetic risk factor for cerebro-cardiovascular disease, especially cardiovascular events, in hypertensive patients in Japan.

Klotho gene delivery suppresses oxidative stress in vivo

Objective:  Mice deficient in the klotho gene exhibit a syndrome resembling premature human aging. A recent report also suggested that klotho transgenic mice exhibited a long lifespan, which shows that klotho is an antisenescence gene. Previously, klotho has been reported to improve endothelial dysfunction, and also to have a preventive effect against oxidative stress. In the present study, we investigated the effect of klotho gene delivery on blood pressure and oxidative stress in vivo.

Relationship between renal hemodynamic status and aging in patients without diabetes evaluated by renal Doppler ultrasonography.

BackgroundAging is well known as one of the major causes of a reduced glomerular filtration rate (GFR). The resistive index (RI) measured by renal Doppler ultrasonography (RDU) is thought to be a good indicator of renal vascular resistance induced by arteriosclerosis. In this study, we investigated whether RI could be used to evaluate the pathogenesis of renal damage or the mechanisms of reduction of renal function by aging.MethodsWe investigated the correlation between RI and multiple clinical parameters and the influence of aging on the renal hemodynamic status of 194 in-patients (mean age 66.2 years) who underwent RDU at our hospital between February 2009 and July 2010.ResultsRI was significantly correlated with the age, estimated GFR (eGFR), diastolic blood pressure, pulse pressure, and degree of albuminuria. Subjects aged ≥75 years showed a significantly higher correlation coefficient between eGFR and RI. RI showed a stronger correlation with age in subjects aged ≥75 years compared to eGFR.ConclusionThe present study showed that renal vascular resistance and intra-renal arteriosclerosis had a greater impact on renal function in older than younger subjects, reflecting the possible mechanisms of renal function reduction due to aging.