Medial artery calcification, which does not accompany lipid or cholesterol deposit, preferentially occurs in elderly population, but its underlying mechanisms remain unclear. In the present study, we investigated the potential role of senescent vascular smooth muscle cells (VSMCs) in the formation of senescence-associated medial calcification. Replicative senescence was induced by the extended passages (until passages 11-13) in human primary VSMCs, and cells in early passage (passage 6) were used as control young cells. VSMC calcification was markedly enhanced in the senescent cells compared with that in the control young cells. We identified that genes highly expressed in osteoblasts, such as alkaline phosphatase (ALP) and type I collagen, were significantly upregulated in the senescent VSMCs, suggesting their osteoblastic transition during the senescence. Knockdown of either ALP or type I collagen significantly reduced the calcification in the senescent VSMCs. Of note, runt-related transcription factor-2 (RUNX-2), a core transcriptional factor that initiates the osteoblastic differentiation, was also upregulated in the senescent VSMCs. Knockdown of RUNX-2 significantly reduced the ALP expression and calcification in the senescent VSMCs, suggesting that RUNX-2 is involved in the senescence-mediated osteoblastic transition. Furthermore, immunohistochemistry of aorta from the klothoaging mouse model demonstrated in vivo emergence of osteoblastlike cells expressing RUNX-2 exclusively in the calcified media. We also found that statin and Rho-kinase inhibitor effectively reduced the VSMC calcification by inhibiting Pi-induced apoptosis and potentially enhancing matrix Gla protein expression in the senescent VSMCs. These findings strongly suggest an important role of senescent VSMCs in the pathophysiology of senescence-associated medial calcification, and the inhibition of osteoblastic transition could be a new therapeutic approach for the prevention of senescence-associated medial calcification.runt-related transcription factor-2; statin VASCULAR CALCIFICATION is widespread in patients with coronary artery disease and peripheral artery disease (21) and is closely associated with the incidence of cardiovascular events as well as all-cause mortality (3,27,34). Calcification in the tunica media is often observed in elderly people and is highly correlated with their morbidity and mortality (8).Many recent findings have suggested that vascular calcification is regulated by the machinery similar to bone formation, which is accomplished through the extracellular matrix (ECM) calcification (16,21,34). During the ECM calcification, hydroxyapatite crystals that contain calcium and inorganic phosphate precipitate within the collagen fibrils (32). Many key players in the ECM calcification, such as matrix Gla protein (MGP) and alkaline phosphatase (ALP), have been identified (22,32). Inorganic pyrophosphate, a small molecule made of two phosphate ions, and MGP prevent incorporation of mineral crystals into the collagen fibrils ...

No abstract

Objective-Vascular calcification is an important risk factor for cardiovascular diseases. Here, we investigated a role of dedifferentiated vascular smooth muscle cells (VSMCs) in the atherosclerotic intimal calcification. Methods and Results-We prepared human cultured VSMCs in either redifferentiatiated or dedifferentiated state and analyzed the gene expressions of bone-calcification regulatory factors. Expression of bone morphogenetic protein-2 (BMP-2), a potent initiator for osteoblast differentiation, was significantly enhanced in dedifferentiated VSMCs. Furthermore, endogenous BMP-2 antagonists, such as noggin, chordin, and matrix gamma-carboxyglutamic acid protein, were all downregulated in the dedifferentiated VSMCs. Conditioned medium from dedifferentiated VSMCs, but not from redifferentiated VSMCs, stimulated the osteoblastic differentiation of the mesenchymal progenitor C2C12 cells, which was abolished by BMP-2 knockdown. In atherosclerotic intima from apolipoprotein (apo)E-deficient mice, ␣SM-actin-positive cells, presumably dedifferentiated VSMCs, expressed BMP-2. We generated BMP-2-transgenic mice using ␣SM-actin promoter and crossed them with apoE-deficient mice (BMP-2-transgenic/apoE-knockout).Significantly accelerated atherosclerotic intimal calcification was detected in BMP-2-transgenic/apoE-knockout mice, although serum lipid concentration and atherosclerotic plaque size were not different from those in apoE-knockout mice. Enhanced calcification appeared to be associated with the frequent emergence of osteoblast-like cells in atherosclerotic intima in BMP-2-transgenic/apoE-knockout mice. V ascular calcification has been an important risk factor for cardiovascular diseases as well as all-causal mortality. 1,2 There are several types of vascular calcification, such as calcification in intima associated with atherosclerosis and calcification in tunica media (medial calcification), which is often observed in elderly people and patients with diabetes mellitus and/or chronic kidney disease. 3 Recently, we have reported an important role of senescent vascular smooth muscle cells (VSMCs) in the formation of medial calcification associated with aging. 4 However, the molecular mechanism(s) governing atherosclerotic intimal calcification remains to be elucidated. Atherosclerotic calcification of coronary artery is a significant risk for the unsuccessful coronary intervention and balloon-induced coronary artery dissection. 5 Calcification score of coronary arteries assessed by electron beam computer tomography has been reported to correlate well with the incidence of cardiovascular diseases. 6 Furthermore, calcification was found to be a reliable marker of plaque instability, defined as plaques that have undergone rupture using autopsy specimens. 7 However, there is a controversy around this with arguments describing calcification as a marker of plaque stability as well. Negative correlation between extensive calcification and plaque instability was shown, and the pattern of calcification rather than the vo...

SummaryThe NSY (Nagoya-Shibata-Yasuda) mouse was established as an inbred strain of mouse with spontaneous development of diabetes mellitus, by selective breeding for glucose intolerance from outbred Jcl:ICR mice. NSY mice spontaneously develop diabetes mellitus in an age-dependent manner. The cumulative incidence of diabetes is 98 % in males and 31% in females at 48 weeks of age. Neither severe obesity nor extreme hyperinsulinaemia is observed at any age in these mice. Glucose-stimulated insulin secretion was markedly impaired in NSY mice after 24 weeks of age. In contrast, fasting plasma insulin level was higher in male NSY mice than that in male C3H/He mice (545 +73 vs 350+ 40 pmol/1, p < 0.05, at 36 weeks of age). Pancreatic insulin content was higher in male NSY mice than that in male C3H/He mice (76 + 8 vs 52 _+ 5 ng/mg wet weight, p < 0.05, at 36 weeks of age). Morphologically, no abnormal findings, such as hypertrophy or inflammatory changes in the pancreatic islets, were observed in NSY mice at any age. These data suggest that functional changes of insulin secretion in response to glucose from pancreatic beta cells may contribute to the development of non-insulin-dependent diabetes mellitus (NIDDM) in the NSY mouse. Although insulin sensitivity was not measured, fasting hyperinsulinaemia in NSY mice suggests that insulin resistance may also contribute to the pathogenesis of NIDDM. Since these findings are similar to the pathophysiologic features of human NIDDM patients, the NSY mouse is considered to be useful for investigating the pathogenesis and genetic predisposition to NIDDM. [Diabetologia (1995) 38: 503-508] Key words NSY mouse, non-insulin-dependent diabetes mellitus, animal model, insulin secretion, isolated islets.Non-insulin-dependent diabetes mellitus (NIDDM) is a heterogeneous disorder, caused by an interaction of genetic and environmental factors [1][2][3]. This heterogeneity in human NIDDM makes it difficult to clarify the genetics or pathogenesis of the disease. Animal models are invaluable for the analysis of heterogeneous disorders such as diabetes. This is eviReceived: 22 June 1994 and in revised form: 11 October 1994 Corresponding author: Dr. H. Ikegami, Department of Geriatric Medicine, Osaka University Medical School, 2-2 Yamadaoka, Suita, Osaka 565, Japan Abbreviations: NIDDM, Non-insulin-dependent diabetes mellitus; IDDM, insulin-dependent diabetes mellitus; NSY mouse, Nagoya-Shibata-Yasuda mouse.denced by the recent progress in the understanding of the genetics and pathogenesis of insulin-dependent diabetes mellitus by use of excellent animal models, such as the nonobese diabetic (NOD) mouse and the Bio-Breeding (BB) rat [4]. Several animal models for NIDDM have been described. Although recent studies have revealed impaired insulin secretion in GK rats [5][6][7], most of the animal models for NIDDM are characterized by obesity, hyperinsulinaemia and islet hypertrophy [8].The NSY (Nagoya-Shibata-Yasuda) mouse is a spontaneous model of NIDDM with moderate obesity that was establ...

A possible pathogenic mutation in the beta 3-adrenergic-receptor gene (Trp64Arg) has been reported to be associated with an earlier age of onset of non-insulin-dependent diabetes mellitus (NIDDM) and clinical features of the insulin resistance syndrome in Pima Indian, Finnish and French subjects. Since marked heterogeneity has been reported in the association of mutations of candidate genes with NIDDM between Japanese and other ethnic groups, we investigated the association of Trp64Arg with NIDDM in Japanese subjects. The allele frequency of the mutation (Arg) was slightly, but not significantly, higher in NIDDM than in control subjects (70 out of 342 alleles [20.5%] vs 40 out of 248 [16.1%], respectively, p > 0.2). When our data were combined with those of Pima Indian and Finnish subjects, however, the Arg/Arg genotype was significantly associated with NIDDM as compared with the other two genotypes (p < 0.005, relative risk [RR] 2.13, 95% confidence interval [CI] 1.28-3.55). The Arg allele was also associated with NIDDM (p < 0.05, RR 1.27, 95% CI 1.06-1.52). Japanese subjects homozygous for the mutation had a significantly higher body mass index (mean +/- SD: 25.5 +/- 3.9 kg/m2) than heterozygotes (22.6 +/- 4.1, p < 0.05) and normal homozygotes (22.8 +/- 3.8, p < 0.05). NIDDM patients homozygous for the mutation tended to have an earlier age of onset of NIDDM than those with other genotypes. These data suggest that the Trp64Arg mutation not only contributes to weight gain and age-at-onset of NIDDM but is also associated with susceptibility to NIDDM.

The evolution of different forms of photosynthetic life has profoundly altered the activity level of the biosphere, radically reshaping the composition of Earth's oceans and atmosphere over time. However, the mechanistic impacts of a primitive photosynthetic biosphere on Earth's early atmospheric chemistry and climate are poorly understood. Here, we use a global redox balance model to explore the biogeochemical and climatological effects of different forms of primitive photosynthesis. We find that a hybrid ecosystem of H2-based and Fe 2+ -based anoxygenic photoautotrophs-organisms that perform photosynthesis without producing oxygen-gives rise to a strong nonlinear amplification of Earth's methane (CH4) cycle, and would thus have represented a critical component of Earth's early climate system before the advent of oxygenic photosynthesis. Using a Monte Carlo approach, we find that a photosynthetic hybrid biosphere widens the range of geochemical conditions that allow for warm climate states well beyond either of these metabolisms acting in isolation. Our results imply that the Earth's early climate was governed by a novel and poorly explored set of regulatory feedbacks linking the anoxic biosphere and the coupled H, C, and Fe cycles, with important ramifications for the sustained habitability of reducing Earth-like planets hosting primitive photosynthetic life.

Background The female preponderance in heart failure with preserved ejection fraction (HFpEF) is a distinguishing feature of this disorder, but the association of sex with degree of diastolic dysfunction and clinical outcomes among individuals with HFpEF remains unclear. Methods and Results We conducted a prospective, multicenter, observational study of patients with HFpEF (PURSUIT‐HFpEF [Prospective Multicenter Observational Study of Patients with Heart Failure with Preserved Ejection Fraction]: UMIN000021831). Between 2016 and 2019, 871 patients were enrolled from 26 hospitals (follow‐up: 399±349 days). We investigated sex‐related differences in diastolic dysfunction and postdischarge clinical outcomes in patients with HFpEF. The echocardiographic end point was diastolic dysfunction according to American Society of Echocardiography/European Association of Cardiovascular Imaging criteria. The clinical end point was a composite of all‐cause death and heart failure readmission. Women accounted for 55.2% (481 patients) of the overall cohort. Compared with men, women were older and had lower prevalence rates of hypertension, coronary artery disease, and chronic kidney disease. Women had diastolic dysfunction more frequently than men (52.8% versus 32.0%, P <0.001). The incidence of the clinical end point did not differ between women and men (women 36.1/100 person‐years versus men 30.5/100 person‐years, P =0.336). Female sex was independently associated with the echocardiographic end point (adjusted odds ratio, 2.839; 95% CI, 1.884–4.278; P <0.001) and the clinical end point (adjusted hazard ratio, 1.538; 95% CI, 1.143–2.070; P =0.004). Conclusions Female sex was independently associated with the presence of diastolic dysfunction and worse clinical outcomes in a cohort of elderly patients with HFpEF. Our results suggest that a sex‐specific approach is key to investigating the pathophysiology of HFpEF. Registration URL: https://upload.umin.ac.jp ; Unique identifier: UMIN000021831.

Patterns of linkage disequilibrium (LD) in the human genome are beginning to be characterized, with a paucity of haplotype diversity in "LD blocks," interspersed by apparent "hot spots" of recombination. Previously, we cloned and physically characterized the low-density lipoprotein-receptor-related protein 5 (LRP5) gene. Here, we have extensively analysed both LRP5 and its flanking three genes, spanning 269 kb, for single nucleotide polymorphisms (SNPs), and we present a comprehensive SNP map comprising 95 polymorphisms. Analysis revealed high levels of recombination across LRP5, including a hot-spot region from intron 1 to intron 7 of LRP5, where there are 109 recombinants/Mb (4882 meioses), in contrast to flanking regions of 14.6 recombinants/Mb. This region of high recombination could be delineated into three to four hot spots, one within a 601-bp interval. For LRP5, three haplotype blocks were identified, flanked by the hot spots. Each LD block comprised over 80% common haplotypes, concurring with a previous study of 14 genes that showed that common haplotypes account for at least 80% of all haplotypes. The identification of hot spots in between these LD blocks provides additional evidence that LD blocks are separated by areas of higher recombination.