Omentin inhibits osteoblastic differentiation of calcifying vascular smooth muscle cells through the PI3K/Akt pathway

Arterial calcification is positively associated with visceral adiposity, but the mechanisms remain unclear. Omentin is a novel adipokine that is selectively expressed in visceral adipose tissue. The levels of circulating omentin are decreased in obesity, and they correlate negatively with waist circumference. This study investigated the effects of omentin on the osteoblastic differentiation of calcifying vascular smooth muscle cells (CVSMCs), a subpopulation of aortic smooth muscle cells putatively involved in vascular calcification. Omentin inhibited mRNA expression of alkaline phosphatase (ALP) and osteocalcin; omentin also suppressed ALP activity, osteocalcin protein production, and the matrix mineralization. Furthermore, omentin selectively activated phosphatidylinositol 3-kinase (PI3K) downstream effector Akt. Moreover, inhibition of PI3K or Akt activation reversed the effects of omentin on ALP activity and the matrix mineralization. The present results demonstrate for the first time that omentin can inhibit osteoblastic differentiation of CVSMCs via PI3K/Akt signaling pathway, suggesting that the lower omentin levels in obese (specially visceral obese) subjects contribute to the development of arterial calcification, and omentin plays a protective role against arterial calcification.     

Abdominal Adipose Tissue was Associated with Glomerular Hyperfiltration among Non- Diabetic and Normotensive Adults with a Normal Body Mass Index

Glomerular hyperfiltration is recognized as an early marker of progressive kidney dysfunction in the obese population. This study aimed to identify the relationship between glomerular hyperfiltration and body fat distribution measured by computed tomography (CT) in healthy Korean adults. The study population included individuals aged 20–64 years who went a routine health check-up including an abdominal CT scan. We selected 4,378 individuals without diabetes and hypertension. Glomerular filtration rate was estimated using the CKD-EPI equation, and glomerular hyperfiltration was defined as the highest quintile of glomerular filtration rate. Abdominal adipose tissue areas were measured at the level of the umbilicus using a 16-detector CT scanner, and the cross-sectional area was calculated using Rapidia 2.8 CT software. The prevalence of glomerular hyperfiltration increased significantly according to the subcutaneous adipose tissue area in men (OR = 1.74 (1.16–2.61), P for trend 0.016, for the comparisons of lowest vs. highest quartile) and visceral adipose tissue area in women (OR = 2.34 (1.46–3.75), P for trend < 0.001) in multivariate analysis. After stratification by body mass index (normal < 23 kg/m², overweight ≥ 23 kg/m²), male subjects with greater subcutaneous adipose tissue, even those in the normal BMI group, had a higher prevalence of glomerular hyperfiltration (OR = 2.11 (1.17–3.80), P for trend = 0.009). Among women, the significance of visceral adipose tissue area on glomerular hyperfiltration resulted from the normal BMI group (OR = 2.14 (1.31–3.49), P for trend = 0.002). After menopause, the odds ratio of the association of glomerular hyperfiltration with subcutaneous abdominal adipose tissue increased (OR = 2.96 (1.21–7.25), P for trend = 0.013). Subcutaneous adipose tissue areas and visceral adipose tissue areas are positively associated with glomerular hyperfiltration in healthy Korean adult men and women, respectively. In post-menopausal women, visceral adipose tissue area shows significant positive association with glomerular hyperfiltration as in men.     

Carbon monoxide effects on calcium levels in vascular smooth muscle

Previously we showed that carbon monoxide (CO) relaxes vascular smooth muscle in the working heart and thoracic aorta preparations perfused with hemoglobin-free, Krebs-Henseleit (KH) solution. The CO-induced relaxation was not caused by hypoxia, nor was it mediated by adrenergic influences, adenosine, or prostaglandins. In these studies the effect of CO on calcium (Ca++) concentrations in vascular smooth muscle was determined using 45Ca as a tracer. Isolated rat thoracic aorta segments were incubated with 45Ca and gassed with O2, N2, or CO for 60 min. Verapamil was used to verify the effectiveness of the test system. Ca++ concentrations were 488 +/- 35 and 515 +/- 26 mM/g tissue (X +/- SE) in aortic rings gassed with O2 and N2, respectively. CO reduced Ca++ concentrations significantly (P less than 0.01) by 29% to 369 +/- 18 mM/g tissue. Verapamil treatment reduced Ca++ concentrations by 40% to 314 +/- 23 mM/g tissue. These results suggest that CO relaxes vascular smooth muscle and dilates blood vessels by decreasing Ca++ concentrations in vascular smooth muscle.     

Higher Free Fatty Acid Uptake in Visceral Than in Abdominal Subcutaneous Fat Tissue in Men

Visceral adipose tissue has been shown to have high lipolytic activity. The aim of this study was to examine whether free fatty acid (FFA) uptake into visceral adipose tissue is enhanced compared to abdominal subcutaneous tissue in vivo. Abdominal adipose tissue FFA uptake was measured using positron emission tomography (PET) and [¹⁸F]‐labeled 6‐thia‐hepta‐decanoic acid ([¹⁸F]FTHA) and fat masses using magnetic resonance imaging (MRI) in 18 healthy young adult males. We found that FFA uptake was 30% higher in visceral compared to subcutaneous adipose tissue (0.0025 ± 0.0018 vs. 0.0020 ± 0.0016 µmol/g/min, P = 0.005). Visceral and subcutaneous adipose tissue FFA uptakes were strongly associated with each other (P < 0.001). When tissue FFA uptake per gram of fat was multiplied by the total tissue mass, total FFA uptake was almost 1.5 times higher in abdominal subcutaneous than in visceral adipose tissue. In conclusion, we observed enhanced FFA uptake in visceral compared to abdominal subcutaneous adipose tissue and, simultaneously, these metabolic rates were strongly associated with each other. The higher total tissue FFA uptake in subcutaneous than in visceral adipose tissue indicates that although visceral fat is active in extracting FFA, its overall contribution to systemic metabolism is limited in healthy lean males. Our results indicate that subcutaneous, rather than visceral fat storage plays a more direct role in systemic FFA availability. The recognized relationship between abdominal visceral fat mass and metabolic complications may be explained by direct effects of visceral fat on the liver.     

Indoleamine 2,3-diooxygenase in periaortic fat: mechanisms of inhibition of contraction

Indoleamine 2,3-dioxygenase (IDO) metabolizes L-tryptophan to L-kynurenine, promotes immunosuppression, and has been described as a consumer of superoxide. We discovered IDO expression in periaortic fat and tested the hypothesis that periarterial IDO functionally reduces agonist-induced contraction. Our model was the thoracic aorta, abdominal aorta, and superior mesenteric artery of the male Sprague-Dawley rat. Periaortic fat from the thoracic aorta stained intensely for IDO, the brown fat marker uncoupling protein-1, and oil red O as a general lipid marker. White fat around the mesenteric artery and abdominal aorta stained less for IDO; brown fat was less abundant. IDO activity (kynurenine-to-tryptophan ratio via HPLC) was detected in visceral and mesenteric artery fat (ratio: ～4) but was highest in perithoracic aortic fat (ratio: 10 ± 1.1). In isometric contractile experiments, periadventitial fat reduced ANG II-induced thoracic aortic (with fat: 34% of without fat) and mesenteric artery (with fat: 63% of without fat) maximal contraction. In contrast, periadventitial fat did not reduce agonist-induced contraction in the abdominal aorta. The IDO inhibitor 1-L-methyltryptophan (1-MT) reversed the fat-induced reduction of ANG II-induced contraction in the thoracic aorta but not in the mesenteric artery. The IDO metabolite kynurenine relaxed the thoracic aorta only at high (9 mM) concentrations, whereas the downstream metabolite quinolinic acid (1 mM) relaxed the contracted thoracic aorta (～80%). 1-MT did not correct the reduction in basal superoxide levels observed in the presence of perithoracic aortic fat. We conclude that IDO is an enzyme active primarily in brown fat surrounding the thoracic aorta and depresses aortic contractility.     

Circulating S100A12 Levels Are Associated with Progression of Abdominal Aortic Calcification in Hemodialysis Patients

Vascular calcification is an important factor associated with mortality in dialysis patients. Recently, soluble receptor for advanced glycation end product (sRAGE) and extracellular RAGE binding protein S100A12 (EN-RAGE) have been reported to be involved in the process of vascular calcification. Therefore, we investigated whether sRAGE and S100A12 are useful indicators of progression of abdominal aortic calcification in hemodialysis (HD) patients. We analyzed annual changes in vascular calcification score (VCS) for up to 4 years, compared to clinical and biological parameters in 149 HD patients. VCS was assessed annually using plain X-ray images of the lateral lumbar spine. The progression group was defined as patients with an increase in VCS more than 1 point each year on average during the observation period. Time-averaged concentrations were also evaluated to examine the association between biological parameters and changes in VCS. The patients had a mean age of 58.59 ± 12.93 years; 53.7% were male, and 45% were diabetic. The VCS increased in 55 patients; the mean increase was 1.60 ± 2.91 points. In a stepwise multivariate logistic analysis, we found that higher levels of S100A12 were significantly associated with progression of VCS (odds ratio [OR], 2.622; 95% confidence interval [CI], 1.371–5.016; P = 0.004). The relationship between sRAGE and VCS was not statistically significant (OR, 0.644; 95% CI, 0.302–1.374; P = 0.255). Our findings suggest that serum levels of S100A12 are associated with progression of abdominal aortic calcification in HD patients, independent of sRAGE level.     

Interleukin-1beta causes different levels of nitric oxide-mediated depression of contractility in different positions of rat thoracic aorta.

Interleukin-1beta (IL-1beta) can be synthesized by macrophages, endothelial cells and vascular smooth muscle cells when stimulated by bacterial lipopolysaccharide (endotoxin) during septic shock. The IL-1beta levels in the blood vessel wall are also elevated in atherosclerosis. IL-1beta can cause induction of inducible nitric oxide synthase (iNOS) expression in vascular smooth muscle cells and produce vasorelaxation, hypotension and ultimately tissue damage. We studied the depressions of vascular smooth muscle contractions at 3 hours after exposure to IL-1beta in different positions of rat thoracic aorta. The data show that the aortic rings from the cranial end of rat thoracic aorta had little response to IL-1beta (0.5 and 1.0 ng/ml) while those from the caudal end of thoracic aorta had larger depressant response. S-methylisothiourea sulfate (SMT), an iNOS inhibitor, completely blocked the depression of contraction caused by IL-1beta in intact aortic rings. If the endothelium was removed from the aortic rings before exposure to IL-1beta, all rings from different parts of the thoracic aorta showed an equal amount of vasodepression. Thus, the difference in the depressant response of IL-1beta in different portions of thoracic aorta is endothelium-dependent and involves induction of NOS.     

A new selective ultrahistochemical method for the demonstration of calcium using N,N-naphthaloylhydroxylamine

Summary A new, simple, rapid, and highly sensitive and selective method for the ultrahistochemical detection of calcium is described. The reagent N,N-Naphthaloylhydroxylamine (1,8-C₁₀H₆CON(ONa)CO) sodium salt was employed in this study for the demonstration of calcium at the subcellular level in relaxed and contracted muscles (smooth muscle of the stomach, thoracic aorta, and myocardial muscle cells) of the rat (in vitro as well as in vivo) and in the human vascular smooth muscle of the aorta with atherosclerotic calcification.Direct evidence of the presence of calcium in the electron-dense reaction products (calcium N,N-Naphthaloylhydroxylamine) is given by X-ray microanalysis of 1,500–2,000 Å thick sections.The significance of distributional differences in the localization of calcium in subcellular structures of relaxed and contracted muscles in discussed in relation to the role of calcium in the control of the muscle activity during the contraction-relaxation cycle.     

大鼠降主动脉雌激素受体的免疫组织化学研究

冰冻切片微波修复后,用ER单克隆抗体免疫组织化学SP法及图像分析方法,观察雌激素受体（ER）在雌性与雄性大鼠降主动脉壁的分布,结果显示,ER阳性反应存在于血管壁的内皮细胞与平滑肌细胞中,阳性反应强度无性别差异。无段间差异（降主动脉胸段与腹段）,血管周围的脂肪组织也可见大量呈阳性反应的细胞,结果表明,降主动脉壁的内皮细胞,血管平滑肌细胞中存在ER。降主动脉可能是雌激素的靶器官。     

Accumulation of calcium and phosphorus in the mitral valve in comparison with the abdominal aorta and the scaphoid bone

To clarify why calcification of the mitral valve occurred, the authors chose the abdominal aorta and the scaphoid bone among many arteries and bones, and they studied both relationships in element contents between the mitral valve and the abdominal aorta and between the mitral valve and the scaphoid bone. The subjects consisted of 11 men and 8 women, ranging in age from 52 to 96 yr. The accumulation of calcium and phosphorus occurred progressively with aging in the mitral valve, whereas it became the highest in the sixties in the abdominal aorta and did not increase thereafter. The accumulation of calcium and phosphorus occurred in the abdominal aorta earlier than the thoracic aorta, in which it became remarkable in the seventies. It should be noted that in regard to the accumulation of calcium and phosphorus, no significant correlations were found between the mitral valve and the abdominal aorta. It is suggested that calcification of the abdominal aorta is not essentially accompanied by calcification of the mitral valve. The scaphoid bone was chosen among many bones consisting mainly of spongy bone and the relationship was examined between the calcium content in the mitral valve and the bone mineral density of the scaphoid bone. It was found that there was a low relationship between them. Therefore, it is suggested that a part of the surplus calcium released from bones is deposited in the mitral valve.     

Aortic stiffness is associated with vascular calcification and remodeling in a chronic kidney disease rat model

Increased aortic pulse-wave velocity (PWV) reflects increased arterial stiffness and is a strong predictor of cardiovascular risk in chronic kidney disease (CKD). We examined functional and structural correlations among PWV, aortic calcification, and vascular remodeling in a rodent model of CKD, the Lewis polycystic kidney (LPK) rat. Hemodynamic parameters and beat-to-beat aortic PWV were recorded in urethane-anesthetized animals [12-wk-old hypertensive female LPK rats (n = 5)] before the onset of end-stage renal disease and their age- and sex-matched normotensive controls (Lewis, n = 6). Animals were euthanized, and the aorta was collected to measure calcium content by atomic absorption spectrophotometry. A separate cohort of animals (n = 5/group) were anesthetized with pentobarbitone sodium and pressure perfused with formalin, and the aorta was collected for histomorphometry, which allowed calculation of aortic wall thickness, medial cross-sectional area (MCSA), elastic modulus (EM), and wall stress (WS), size and density of smooth muscle nuclei, and relative content of lamellae, interlamellae elastin, and collagen. Mean arterial pressure (MAP) and PWV were significantly greater in the LPK compared with Lewis (72 and 33%, respectively) animals. The LPK group had 6.8-fold greater aortic calcification, 38% greater aortic MCSA, 56% greater EM/WS, 13% greater aortic wall thickness, 21% smaller smooth muscle cell area, and 20% less elastin density with no difference in collagen fiber density. These findings demonstrate vascular remodeling and increased calcification with a functional increase in PWV and therefore aortic stiffness in hypertensive LPK rats.     

Spontaneous Aortic Lesions in Fallow Deer (Dama Dama L)

In 19 out of 22 aortas from fallow deer, 15 months to 5½ years, aortic lesions were found. Three types of lesions were observed, fatty streaks were seen in 2, fibrous plaques in 15 and fibrous plaques with complicated lesions (calcification and acid mucopolysaccharides) in 2 of the aortas. Elastic tissue degeneration of the inner two thirds of the tunica media was frequently found in the aorta of the animals > 3½–5½ years of age. There was no statistical evidence for a correlation between age and frequency (P ~ 0.10) but a trend towards age dependence was seen. The percentage of involved surface was found to significantly increase (0.05 > P > 0.01) with age. Lesions were found to start in the abdominal aorta in young animals and to extend cranially to the thoracic aorta with age. The percentage of involved surface in different affected regions, comprising all age groups were, in the posterior abdominal portion, 10.5 %, in the anterior abdominal portion, 4.3 % and in the posterior thoracic portion, 1.04 %. The influence of hemodynamic flow upon the localization of the aortic lesions, the endothelial cell population density and the endothelial nuclear patterns were discussed.     

Reactivity of rat abdominal aorta to U46619 following whole-body gamma irradiation

Rats exposed to 20 Gy whole-body irradiation demonstrated a depressed aortic responsiveness to the thromboxane mimic, U46619, 48 h postirradiation. The mechanism for this observed response was investigated. Shielding the abdominal aorta attenuated this altered vascular reactivity. Since this suggests that radiation exposure induces local changes in the aorta, vascular smooth muscle function was assessed with cumulative concentrations of KCl. Radiation-induced smooth muscle damage was insufficient to account for the decreased reactivity to U46619. Next, calcium availability for vascular smooth muscle function was evaluated and found not to be responsible for the radiation-induced depression in aortic responsiveness. Finally, the role that cyclooxygenase products play in the depressed contractile response was investigated. Indomethacin treatment prior to and for 48 h after irradiation attenuated the altered vascular reactivity to U46619. These data suggest that a radiation-induced increase in cyclooxygenase products may play a role in the decreased aortic reactivity to the thromboxane mimic.     

Calcifications of the Thoracic Aorta on Extended Non-Contrast-Enhanced Cardiac CT

Background

The presence of calcified atherosclerosis in different vascular beds has been associated with a higher risk of mortality. Thoracic aorta calcium (TAC) can be assessed from computed tomography (CT) scans, originally aimed at coronary artery calcium (CAC) assessment. CAC screening improves cardiovascular risk prediction, beyond standard risk assessment, whereas TAC performance remains controversial. However, the curvilinear portion of the thoracic aorta (TA), that includes the aortic arch, is systematically excluded from TAC analysis. We investigated the prevalence and spatial distribution of TAC all along the TA, to see how those segments that remain invisible in standard TA evaluation were affected.

Methods and Results

A total of 970 patients (77% men) underwent extended non-contrast cardiac CT scans including the aortic arch. An automated algorithm was designed to extract the vessel centerline and to estimate the vessel diameter in perpendicular planes. Then, calcifications were quantified using the Agatston score and associated with the corresponding thoracic aorta segment. The aortic arch and the proximal descending aorta, “invisible” in routine CAC screening, appeared as two vulnerable sites concentrating 60% of almost 11000 calcifications. The aortic arch was the most affected segment per cm length. Using the extended measurement method, TAC prevalence doubled from 31% to 64%, meaning that 52% of patients would escape detection with a standard scan. In a stratified analysis for CAC and/or TAC assessment, 111 subjects (46% women) were exclusively identified with the enlarged scan.

Conclusions

Calcium screening in the TA revealed that the aortic arch and the proximal descending aorta, hidden in standard TA evaluations, concentrated most of the calcifications. Middle-aged women were more prone to have calcifications in those hidden portions and became candidates for reclassification.     

Adipocyte triglyceride lipase expression in human obesity

We have investigated the gene and protein expression of adipose triglyceride lipase (ATGL) and triglyceride (TG) lipase activity from subcutaneous and visceral adipose tissue of lean and obese subjects. Visceral and subcutaneous adipose tissue was obtained from 16 age-matched lean and obese subjects during abdominal surgery. Tissues were analyzed for mRNA expression of lipolytic enzymes by real-time quantitative PCR. ATGL protein content was assessed by Western blot and TG lipase activity by radiometric assessment. Subcutaneous and visceral adipose tissue of obese subjects had elevated mRNA expression of PNPLA2 (ATGL) and other lipases including PNPLA3, PNPLA4, CES1, and LYPLAL1 (P < 0.05). Surprisingly, ATGL protein expression and TG lipase activity were reduced in subcutaneous adipose tissue of obese subjects. Immunoprecipitation of ATGL reduced total TG lipase activity in adipose lysates by 70% in obese and 83% in lean subjects. No significant differences in the ATGL activator CGI-58 mRNA levels (ABHD5) were associated with obesity. These data demonstrate that ATGL is important for efficient TG lipase activity in humans. They also demonstrate reduced ATGL protein expression and TG lipase activity despite increased mRNA expression of ATGL and other novel lipolytic enzymes in obesity. The lack of correlation between ATGL protein content and in vitro TG lipase activity indicates that small decrements in ATGL protein expression are not responsible for the reduction in TG lipase activity observed here in obesity, and that posttranslational modifications may be important.     

Spontaneous Aortic Lesions in Moose (Alces Alces L)

In 17 out of 22 aortas from moose, 6 months to more than 18 years, aortic lesions were found. Five different types of lesions were observed, fatty streaks were seen in 1, fibrous plaques in 7, fatty streaks with complicated lesions (calcification and acid mucopolysaccharides) in 3, fibrous plaques with complicated lesions in 2 and fibrous plaques with complicated lesions and fat droplets in 4 of the aortas. Elastic tissue degeneration of the inner two thirds of the tunica media was principally found in the aorta of the animals beyond 4 year of age. There was no statistical evidence for a correlation between age and frequency but a trend towards age dependence was seen. The percentage of involved surface was found to increase significantly (0.05 > P > 0.02) with age. There was no significant difference between affected male and female animals in the different age groups. The frequency of involved surface in different affected regions, comprising all age groups were, in the posterior abdominal portion, 13.0 %, in the anterior abdominal portion, 3.0 %, in the posterior thoracic portion 5.2 %, in the anterior thoracic portion, 0.1 % and in the aortic arch, 0.1 %. Proliferating endothelial cells and the source of smooth muscle cells were discussed.     

High circulating levels of S100A8/A9 complex (calprotectin) in male Japanese with abdominal adiposity and dysregulated expression of S100A8 and S100A9 in adipose tissues of obese mice

S100A8/A9 complex, calprotectin, which serves as an endogenous ligand for immune pathways, is associated with atherosclerosis. These proteins are reported to have several functions such as activating NADPH oxidase, binding toll-like receptor 4 and associated with the receptor for advanced glycation end-products. We recently reported S100A8 mRNA was highly expressed in mouse white adipose tissues and differentiated 3T3-L1 adipocytes. However, regulation of S100A9 expression in murine adipose tissue remains to be elucidated. The results of our studies in male Japanese, obese and control mice and cultured cells showed: (1) serum levels of S100A8/A9 complex, calprotectin, correlated with visceral fat area, body mass index, subcutaneous fat area, and leukocyte count in 500 Japanese men, and (2) higher mRNA expression levels of S100A8 in mature adipocyte fraction and S100A9 in stromal vascular cell fraction of obese mice, compared with those of lean mice. Overexpression of S100A8 and S100A9 in obese adipose tissue may be involved, at least partly, in not only high circulating levels of S100A8/A9 complex in abdominal obesity but also adipose and systemic tissue inflammation.