Endothelin-1 is a potent regulator in vivo in vascular calcification and in vitro in calcification of vascular smooth muscle cells

We observed changes of endothelin content and endothelin mRNA in vivo in vascular calcification and in vitro in calcification of vascular smooth muscle cells to explore the role of endothelin in vascular calcification. Calcification model in vivo was induced by administration of Vitamin D(3) plus nicotine. Calcification of vascular smooth muscle cells (VSMCs) was induced by beta-glycerophosphate. Endothelin content was measured by using radioimmunoassay. Endothelin mRNA amount was determined by using competitive quantitative RT-PCR. The results showed that calcium content, 45Ca(2+) uptake and alkaline phosphatase (ALP) activity were increased in calcified VSMCs, compared with controls, but were decreased, compared with calcified VSMCs plus BQ123 group. The endothelin content in the medium and endothelin mRNA in VSMCs were elevated by 35 and 120% (P<0.05), respectively, compared with those normal VSMCs. Calcium content, 45Ca(2+) accumulation and ALP activity in calcified arteries increased by 5.0-, 1.4-, and 1.4-fold. The endothelin levels in plasma and aorta as well as the amount of endothelin mRNA in calcified aorta were increased by 102, 103, and 22%, respectively, compared with control group. However, calcium content, 45Ca(2+) uptake and ALP activity in VDN plus bosentan group was 33, 36.7, and 40.4% lower than those in VDN group. These results indicated an upregulated endothelin gene expression as well as an increased production of endothelin in calcified aorta and VSMCs with BQ123 and bosentan significantly reducing vascular calcification. This suggested that endothelin might be involved in pathogenesis of vascular calcification.     

Exosomes,the message transporters in vascular calcification

Vascular calcification (VC) is caused by hydroxyapatite deposition in the intimal and medial layers of the vascular wall, leading to severe cardiovascular events in patients with hypertension, chronic kidney disease and diabetes mellitus. VC occurrences involve complicated mechanism networks, such as matrix vesicles or exosomes production, osteogenic differentiation, reduced cell viability, aging and so on. However, with present therapeutic methods targeting at VC ineffectively, novel targets for VC treatment are demanded. Exosomes are proven to participate in VC and function as initializers for mineral deposition. Secreted exosomes loaded with microRNAs are also demonstrated to modulate VC procession in recipient vascular smooth muscle cells. In this review, we targeted at the roles of exosomes during VC, especially at their effects on transporting biological information among cells. Moreover, we will discuss the potential mechanisms of exosomes in VC.     

Heritability of Body Composition Measured by DXA in the Diabetes Heart Study

Objective: The purpose of this study was to investigate the heritability of body composition measured by DXA in the Diabetes Heart Study (DHS). Research Methods and Procedures: Participants were 292 women and 262 men (age, 38 to 86 years; BMI, 17 to 57 kg/m²) from 244 families. There were 492 white and 49 African‐American sibling pairs. DXA measurements of percentage fat mass (FM), whole body FM, and lean mass (LM), as well as regional measurements of trunk fat mass (TFM) and appendicular lean mass (ALM), were obtained. Heritability of FM, LM, and BMI were estimated using Sequential Oligogenic Linkage Analysis Routines. Results: After adjusting for age, gender, ethnicity, and height, the heritability estimates of various compositional attributes were %FM = 0.64, whole body FM = 0.71, TFM = 0.63, whole body LM = 0.60, ALM = 0.66, and BMI = 0.64 (all p < 0.0001). Additional adjustment for diabetes status, smoking, dietary intake, and physical activity resulted in only minor changes in the heritability estimates (?² = 0.63 to 0.72, all p < 0.0001). Furthermore, heritability of TFM after additional adjustment for whole body FM was significant (?² = 0.55, p < 0.0001), and heritability of ALM after additional adjustment for whole body LM was also significant (?² = 0.51, p < 0.0001). Discussion: These data suggest that FM and LM measured by DXA are highly heritable and can be effectively used in designing linkage studies to locate genes governing body composition. In addition, regional distribution of FM and LM may be genetically determined.     

Recent advances in multifactorial regulation of vascular calcification

Calcification presents important clinical implications in cardiovascular diseases, especially in coronary arteries. Epidemiological evidence has shown the coexistence of vascular calcification with both atherosclerosis and osteoporosis, and increasing evidence has shown the role of hyperlipidemia and atherogenic phospholipids in vascular calcification. The etiology of vascular calcification is also increasingly recognized as an active process. Vascular calcification initiates with matrix vesicle formation and mineralization following a process similar to that in bone. In addition, many bone regulatory factors have been shown to be present in calcified atherosclerotic lesions. In this review, we focus on the new developments emerging during the past year in regulation of vascular calcification. Regulatory factors include matrix GLA protein, the phosphate cotransporter Pit-1, a calcium-sensing receptor related factor, osteoprotegerin, leptin, bisphosphonates and oxidized lipids. Some of these, including oxidized lipids, osteoprotegerin, and bisphosphonates, appear to regulate mineralization in both bone and vasculature and may account for the co-existence of osteoporosis and atherosclerotic calcification that is independent of age.     

Beneficial role of the phytoestrogen genistein on vascular calcification

Although soy phytoestrogen are proposed to prevent or improve postmenopausal vascular and bone diseases, the currently available data are controversial and unclear. In this study we evaluated the molecular and biochemical action of genistein on the cellular events involved in vascular calcification. Rat monocytes, aortic vascular cell and osteoblasts cultures in vitro exposed to Gen were employed. Gen down regulated the expression of cell adhesion molecules involved in stable leukocyte attachment. Using flow cytometry we found that the PE significantly diminished monocyte integrins CD11b, CD11c and CD18 expression either under basal and pro-inflammatory environment. At endothelial level, Gen also reduced Intercellular Adhesion Molecule 1 mRNA expression. On vascular muscle cells, the PE markedly reduced cell proliferation and migration. When vascular calcification was studied, muscle cells transdifferentiation into osteoblasts like cells was evaluated. Cells were cultured in osteogenic medium for 21 days. The expression of alkaline phosphatase and the presence of calcified nodules in the extracellular matrix were selected as features of muscle transdifferentiation. Calcified muscle cells exhibited higher levels of alkaline phosphatase activity and enhanced deposition of calcium nodules respect to native cells. Both osteoblastic markers were significantly reduced after Gen treatment. In contrast to this anti-osteogenic action, on bone cells Gen promoted osteoblasts growth, enhanced alkaline phosphatase activity and increased matrix mineralization. Its mitogenic action on osteoblasts directly depends on nitric oxide endothelial production stimulated by the PE. The data presented suppose a beneficial role of Gen on bone and vascular cells, with a cross link between both systems.     

New insight of complement system in the process of vascular calcification

The complement system defences against pathogenic microbes and modulates immune homeostasis by interacting with the innate and adaptive immune systems. Dysregulation, impairment or inadvertent activation of complement system contributes to the pathogenesis of some autoimmune diseases and cardiovascular diseases (CVD). Vascular calcification is the pivotal pathological basis of CVD, and contributes to the high morbidity and mortality of CVD. Increasing evidences indicate that the complement system plays a key role in chronic kidney diseases, atherosclerosis, diabetes mellitus and aging-related diseases, which are closely related with vascular calcification. However, the effect of complement system on vascular calcification is still unclear. In this review, we summarize current evidences about the activation of complement system in vascular calcification. We also describe the complex network of complement system and vascular smooth muscle cells osteogenic transdifferentiation, systemic inflammation, endoplasmic reticulum stress, extracellular matrix remodelling, oxidative stress, apoptosis in vascular calcification. Hence, providing a better understanding of the potential relationship between complement system and vascular calcification, so as to provide a direction for slowing the progression of this burgeoning health concern.     

补镁对大鼠血管钙化的影响

目的:在大鼠血管钙化模型上,观察外源性补充硫酸镁对大鼠血管钙化的影响,以探讨硫酸镁在血管钙化中作用及机制。方法:用维生素D3加尼古丁诱导大鼠血管钙化,von Kossa染色、钙含量测定及碱性磷酸酶活性测定判断血管钙化程度;用半定量RT-PCR方法测定血管钙化标志分子骨桥蛋白mRNA水平;用生物化学方法测定血管一氧化氮(NO)、超氧化物歧化酶(SOD)和丙二醛(MDA)含量。结果:钙化组大鼠血压升高,收缩压较对照组高27%(P<0.05);血管von Kossa染色见血管中膜弹性纤维间可见大量棕黑色颗粒沉积,主动脉钙含量及碱性磷酸酶活性分别较对照高3.9倍和3.4倍(P<0.01),钙化血管组织骨桥蛋白mRNA表达上调40%(P<0.01),血管钙化后可加重血管组织过氧化损伤;而诱导钙化后外源性补充硫酸镁可减轻血管钙化程度,与钙化组比较,低、高剂量硫酸镁组均明显缓解上述指标的变化。结论:诱导血管钙化后外源性补充硫酸镁可以减轻大鼠血管钙化和血管损伤程度。     

Biochemical markers of vascular calcification in elderly hemodialysis patients

The increased vascular calcification, cardiovascular morbidity, and mortality in chronic kidney disease (CKD) patients has been associated with disturbances in mineral-bone metabolism. In order to determine markers of the vascular calcification frequently observed in these patients, blood samples of elderly male and female hemodialysis CKD patients were used to measure serum levels of: osteoprotegerin (OPG), total soluble receptor activator of nuclear factor-κB ligand (sRANKL), and fetuin-A by enzyme immunoassay; tartrate-resistant acid phosphatase (TRACP-5b), and bone-specific alkaline phosphatase (BAP) by immunoenzymometric assay; osteocalcin (OC) by ELISA; iPTH by immunoradiometric assay; 25(OH)D₃ and 1,25(OH)₂D₃, by I¹²⁵ radioimmunoassay; and calcium and phosphorus by photometric assay. Serum OPG, BAP, iPTH, phosphorus, and OC levels were higher and serum 25(OH)D₃, 1,25(OH)₂D₃, and fetuin-A levels lower in both male and female CKD patients than in their respective controls. Our results indicate that the bone formation and resorption parameters are altered in elderly male and female hemodialysis CKD patients. These changes may lead to vascular calcifications and cardiovascular complications, given that elevated OPG and OC levels and reduced fetuin-A levels are associated with cardiovascular events.     

Role of cellular cholesterol metabolism in vascular cell calcification

Vascular calcification impairs vessel compliance and increases the risk of cardiovascular events. We found previously that liver X receptor agonists, which regulate intracellular cholesterol homeostasis, augment PKA agonist- or high phosphate-induced osteogenic differentiation of vascular smooth muscle cells. Because cholesterol is an integral component of the matrix vesicles that nucleate calcium mineral, we examined the role of cellular cholesterol metabolism in vascular cell mineralization. The results showed that vascular smooth muscle cells isolated from LDL receptor null (Ldlr(-/-)) mice, which have impaired cholesterol uptake, had lower levels of intracellular cholesterol and less osteogenic differentiation, as indicated by alkaline phosphatase activity and matrix mineralization, compared with WT cells. PKA activation with forskolin acutely induced genes that promote cholesterol uptake (LDL receptor) and biosynthesis (HMG-CoA reductase). In WT cells, inhibition of cholesterol uptake by lipoprotein-deficient serum attenuated forskolin-induced matrix mineralization, which was partially reversed by the addition of cell-permeable cholesterol. Prolonged activation of both uptake and biosynthesis pathways by cotreatment with a liver X receptor agonist further augmented forskolin-induced matrix mineralization. Inhibition of either cholesterol uptake, using Ldlr(-/-) cells, or of cholesterol biosynthesis, using mevastatin-treated WT cells, failed to inhibit matrix mineralization due to up-regulation of the respective compensatory pathway. Inhibition of both pathways simultaneously using mevastatin-treated Ldlr(-/-) cells did inhibit forskolin-induced matrix mineralization. Altogether, the results suggest that up-regulation of cholesterol metabolism is essential for matrix mineralization by vascular cells.     

高同型半胱氨酸血症促进大鼠血管钙化

目的:在大鼠血管钙化模型上,探讨高同型半胱氨酸血症对血管钙化的影响及其作用机制.方法:用维生素D3加尼古丁诱导大鼠血管钙化模型,并给以高蛋氨酸饮食六周诱导大鼠高同型半胱氨酸血症,用高效液相色谱法检测血浆总同型半胱氨酸(Hcy)水平;采用血管组织vonKossa染色、钙含量测定、碱性磷酸酶(ALP)活性和骨钙素(OC)含量测定以判断血管钙化程度,同时测定血浆脂质共轭烯(Diene键)含量反映其脂质过氧化水平.结果:钙化组大鼠血管yon Kossa染色可见大量黑色颗粒沉积,其血管的钙含量,碱性磷酸酶活性及骨钙素含量分别较对照组增加8.09倍、45.57%和2.81倍(P＜0.01).高蛋氨酸饮食的钙化组大鼠血管内钙含量较单纯钙化组增高了34.29%,而碱性磷酸酶活性及骨钙素含量则较单纯的钙化组降低29.13%和74.69%(P＜0.01).钙化组大鼠血浆脂质共轭烯含量与对照组比较无显著性差异,单纯高蛋氨酸饮食和钙化加高蛋氨酸饮食大鼠血浆脂质共轭烯含量较对照组增加了1.93和2.89倍(P＜0.01),而钙化加高蛋氨酸饮食大鼠血浆脂质共轭烯含量较单纯高蛋氨酸饮食大鼠又增加了32.90%(P＜0.01).结论:高同型半胱氨酸血症可以促进血管的钙化,可能与其所致的脂质过氧化程度增强有关.     

Effect of medial calcification on vascular function in uremia

The contribution of medial calcification to vascular dysfunction in renal failure is unknown. Vascular function was measured ex vivo in control, noncalcified uremic, and calcified uremic aortas from rats with adenine-induced renal failure. Plasma urea was 16 ± 4, 93 ± 14, and 110 ± 25 mg/dl, and aortic calcium content was 27 ± 4, 29 ± 2, and 4,946 ± 1,616 nmol/mg dry wt, respectively, in the three groups. Maximal contraction by phenylephrine (PE) or KCl was reduced 53 and 63% in uremic aortas, and sensitivity to KCl but not PE was increased. Maximal relaxation to acetylcholine was impaired in uremic aortas (30 vs. 65%), and sensitivity to nitroprusside was also reduced, indicating some impairment of endothelium-independent relaxation as well. None of these parameters differed between calcified and noncalcified uremic aortas. However, aortic compliance was reduced in calcified aortas, ranging from 17 to 61% depending on the severity of calcification. We conclude that uremic vascular calcification, even when not severe, significantly reduces arterial compliance. Vascular smooth muscle and endothelial function are altered in renal failure but are not affected by medial calcification, even when severe.     

Aging of mesenchymal stem cells is a root cause of vascular calcification

We hypothesize that aging of mesenchymal stem cells breaking the balance between arterial mineral deposition and resorption results in vascular calcification. It is believed that it is a new idea about revealing pathogenesis of artery calcification and defining a range of novel and exciting therapeutic approaches.     

Predicting all‐cause mortality from basic physiology in the Framingham Heart Study

Using longitudinal data from a cohort of 1349 participants in the Framingham Heart Study, we show that as early as 28–38 years of age, almost 10% of variation in future lifespan can be predicted from simple clinical parameters. Specifically, we found diastolic and systolic blood pressure, blood glucose, weight, and body mass index (BMI) to be relevant to lifespan. These and similar parameters have been well‐characterized as risk factors in the relatively narrow context of cardiovascular disease and mortality in middle to old age. In contrast, we demonstrate here that such measures can be used to predict all‐cause mortality from mid‐adulthood onward. Further, we find that different clinical measurements are predictive of lifespan in different age regimes. Specifically, blood pressure and BMI are predictive of all‐cause mortality from ages 35 to 60, while blood glucose is predictive from ages 57 to 73. Moreover, we find that several of these parameters are best considered as measures of a rate of ‘damage accrual’, such that total historical exposure, rather than current measurement values, is the most relevant risk factor (as with pack‐years of cigarette smoking). In short, we show that simple physiological measurements have broader lifespan‐predictive value than indicated by previous work and that incorporating information from multiple time points can significantly increase that predictive capacity. In general, our results apply equally to both men and women, although some differences exist.     

MiR155 modulates vascular calcification by regulating Akt-FOXO3a signalling and apoptosis in vascular smooth muscle cells

microRNA-155 (miR155) is pro-atherogenic; however, its role in vascular calcification is unknown. In this study, we aim to examine whether miR155 regulates vascular calcification and to understand the underlying mechanism. Quantitative real-time PCR showed that miR155 is highly expressed in human calcific carotid tissue and positively correlated with the expression of osteogenic genes. Wound-healing assay and TUNEL staining showed deletion of miR155 inhibited vascular smooth muscle cell (VSMC) migration and apoptosis. miR155 deficiency attenuated calcification of cultured mouse VSMCs and aortic rings induced by calcification medium, whereas miR155 overexpression promoted VSMC calcification. Compared with wild-type mice, miR155^−/− mice showed significant resistance to vitamin D3 induced vascular calcification. Protein analysis showed that miR155 deficiency alleviated the reduction of Rictor, increased phosphorylation of Akt at S473 and accelerated phosphorylation and degradation of FOXO3a in cultured VSMCs and in the aortas of vitamin D3-treated mice. A PI3K inhibitor that suppresses Akt phosphorylation increased, whereas a pan-caspase inhibitor that suppresses apoptosis reduced VSMC calcification; and both inhibitors diminished the protective effects of miR155 deficiency on VSMC calcification. In conclusion, miR155 deficiency attenuates vascular calcification by increasing Akt phosphorylation and FOXO3a degradation, and thus reducing VSMC apoptosis induced by calcification medium.     

Role of calcium-phosphate deposition in vascular smooth muscle cell calcification

In this work we are studying whether calcium phosphate deposition (CPD) during vascular calcification is a passive or a cell-mediated mechanism. Passive CPD was studied in fixed vascular smooth muscle cells (VSMC), which calcify faster than live cells in the presence of 1.8 mM Ca2(+) and 2 mM P(i). CPD seems to be a cell-independent process that depends on the concentration of calcium, phosphate, and hydroxyl ions, but not on Ca × P(i) concentration products, given that deposition is obtained with 2 × 2 and 4 × 1 Ca × P(i) mM2 but not with 2 × 1 or 1 × 4 Ca × P(i) mM2. Incubation with 4 mM P(i) without CPD (i.e., plus 1 mM Ca) does not induce osteogene expression. Increased expression of bone markers such as Bmp2 and Cbfa1 is only observed concomitantly with CPD. Hydroxyapatite is the only crystalline phase in both lysed and live cells. Lysed cell deposits are highly crystalline, whereas live cell deposits still contain large amounts of amorphous calcium. High-resolution transmission electron microscopy revealed a nanostructure of rounded crystallites of 5-10 nm oriented at random in lysed cells, which is compatible with spontaneous precipitation. The nanostructure in live cells consisted of long fiber crystals, 10-nm thick, embedded in an amorphous matrix. This structure indicates an active role of cells in the process of hydroxyapatite crystallization. In conclusion, our data suggest that CPD is a passive phenomenon, which triggers the osteogenic changes that are involved in the formation of a well organized, calcified crystalline structure.     

Role of gut microbiota-derived metabolites on vascular calcification in CKD

The interaction between gut microbiota and the host has gained widespread concern. Gut microbiota not only provides nutrients from the ingested food but also generates bioactive metabolites and signalling molecules to impact host physiology, especially in chronic kidney disease (CKD). The development of CKD, accompanied by changed diet and medication, alters the gut flora and causes the effect in distant organs, leading to clinical complications. Vascular calcification (VC) is an actively regulated process and a high prevalence of VC in CKD has also been linked to an imbalance in gut microbiota and altered metabolites. In this review, we focused on gut microbiota-derived metabolites involved in VC in CKD and explained how these metabolites influence the calcification process. Correcting the imbalance of gut microbiota and regulating microbiota-derived metabolites by dietary modification and probiotics are new targets for the improvement of the gut-kidney axis, which indicate innovative treatment options of VC in CKD.     

Changes of heme oxygenase-carbon monoxide system in vascular calcification in rats

The aim of the present study was to investigate the change in heme oxygenase (HO)-carbon monoxide (CO)-cyclic guanosine monophosphate (cGMP) pathway in vascular calcification. Vascular calcification model was established in rats by using vitamin D(3) and nicotine. Vascular calcium content, alkaline phosphatase (ALP) activity, HO activity, HbCO formation and content of cGMP in vessels were measured. Immunochemistry (IH) for HO 1 expression and in situ hybridization (ISH) for HO 1 mRNA were observed. Compared to those of control rats, the aortic calcium content and vascular ALP activity in rats of the calcified group (VDN group) were obviously increased, but HO 1 activity, CO concentration and cGMP content in vessels of rats in VDN group were markedly decreased. Expressions of HO-1 protein and mRNA were significantly decreased compared to control rats. Vascular calcification might induce a down regulation in vascular HO-CO-cGMP pathway.