The effects of bisphosphonates on ectopic soft tissue mineralization caused by mutations in the ABCC6 gene

Pseudoxanthoma elasticum (PXE) and generalized arterial calcification of infancy (GACI) are heritable ectopic mineralization disorders. Most cases of PXE and many cases of GACI harbor mutations in the ABCC6 gene. There is no effective treatment for these disorders. We explored the potential efficacy of bisphosphonates to prevent ectopic calcification caused by ABCC6 mutations by feeding Abcc6^−/− mice with diet containing etidronate disodium (ETD) or alendronate sodium trihydrate (AST) in quantities corresponding to 1x, 5x, or 12x of the doses used to treat osteoporosis in humans. The mice were placed on diet at 4 weeks of age, and the degree of mineralization was assessed at 12 weeks by quantitation of the calcium deposits in the dermal sheath of vibrissae, a progressive biomarker of the mineralization, by computerized morphometry of histopathologic sections and by direct chemical assay of calcium. We found that ETD, but not AST, at the 12x dosage, significantly reduced mineralization, suggesting that selected bisphosphonates may be helpful for prevention of mineral deposits in PXE and GACI caused by mutations in the ABCC6 gene, when combined with careful monitoring of efficacy and potential side-effects.     

大鼠动脉钙化诱导过程中炎症小体复合物分子的变化

目的:以SD大鼠动脉钙化为模型,研究钙化发生过程中炎症小体复合物分子的变化。方法:大鼠皮下注射维生素D3诱导动脉钙化,并用HE染色检测动脉钙化的发生,用半定量PCR测定钙化发展过程中细胞内NALP3炎症小体复合物核心蛋白NALP3、ASC和Caspase-1的mRNA水平变化。结果:随着动脉钙化的发展,细胞内NALP3、ASC和Caspase-1的mRNA表达水平逐步增加。结论:构建了大鼠动脉钙化模型,并且发现NALP3炎症小体复合物在动物钙化诱导过程中表达增加,为进一步研究动脉钙化的机制及筛选抗动脉硬化药物打下基础。     

Intracellular precipitation of hydroxyapatite mineral and implications for pathologic calcification

In contrast to physiologic biomineralization occurring in bones, teeth and otoconia in vertebrates, calcification of soft tissues occurs in many pathologic conditions. Although similarities have been noted between the two processes, and despite the important clinical consequences of ectopic calcification, the molecular mechanisms regulating ectopic calcification are poorly understood. Although calcification is mainly extracellular, intracellular calcification has been reported and might indeed contribute to pathologic calcification of soft tissues. To better understand the process of intracellular calcification as a potential origin for pathologic calcification, and to examine the role of proteoglycans in this process, we investigated a pattern of intracellular nucleation and growth of hydroxyapatite in Madin–Darby Canine Kidney (MDCK) epithelial cells using electron microscopy, secondary ion mass spectroscopy (NanoSIMS), cytochemical staining, immunolabeling and biochemical analysis. We report here that under mineralizing cell culture conditions where β-glycerophosphate (βGP) was added as an exogenous organic source of phosphate, βGP-cleaving alkaline phosphatase activity increased and hydroxyapatite crystals subsequently nucleated within intracellular, membrane-bounded compartments. The small, leucine-rich proteoglycan decorin was also upregulated and associated with mineral in these cultures. Such information provides insight into the mechanisms leading to pathologic calcification and describes a process whereby hydroxyapatite deposition in cells might lead to ectopic calcification.     

动脉粥样硬化斑块钙化机制的研究进展

心血管疾病中动脉粥样硬化斑块的钙化是动脉粥样硬化的临床标志之一,主要发生在动脉血管的内膜.动脉粥样硬化斑块核心的钙化不会增加斑块的易损性,而粥样斑块纤维帽上的微钙化会加强纤维帽的周向应力,致使斑块的易损性增加.动脉粥样硬化斑块的钙化机制包括被动钙化和主动钙化,被动钙化受激素和局部信号的调节,主动钙化机制涉及复杂的细胞生命过程,基质囊泡、细胞凋亡、外泌体、氧化应激反应和细胞自噬等均参与了钙化过程.本文对动脉粥样硬化斑块的钙化机制的进展进行综述.     

维持性血液透析患者血清sTWEAK水平与冠状动脉钙化的相关性研究

目的:探讨维持性血液透析患者血清可溶性肿瘤坏死因子样凋亡微弱诱导剂(s TWEAK)水平与冠状动脉钙化的关系。方法:选择维持性血液透析患者60例和健康对照组30例,采用双抗体夹心酶联免疫吸附技术测定其血清s TWEAK水平,采用多层螺旋CT(MSCT)测定其冠状动脉钙化积分(CACs),比较两组的血清s TWEAK水平、CACs,并分析二者的相关性及血液透析患者冠状动脉钙化的危险因素。结果:血液透析患者血清s TWEAK水平显著低于正常对照组,分别为186.23(148.35,220.74)pg/m L和265.13(210.91,298.22)pg/m L,差异有统计学意义(P<0.01)。CACs>400的血液透析患者血清s TWEAK水平显著高于CACs≤400者,分别为220.73(189.70,251.67)pg/m L和146.07(138.43,180.11)pg/m L,差异有统计学意义(P<0.01)。Spearman等级相关分析显示血液透析患者的血清s TWEAK水平与患者的CACs呈明显正相关(r=0.482,P<0.01)。多因素逐步回归分析显示影响血液透析患者CACs严重程度的因素有血清s TWEAK水平、年龄、透析龄、糖尿病(P<0.05)。结论:维持性血液透析患者血清s TWEAK水平较正常人降低,但在血液透析患者s TWEAK范围内,高水平的血清s TWEAK水平与重度冠状动脉钙化相关;血清s TWEAK可能参与了血液透析患者冠状动脉钙化的发生、发展。     

Effects of proteoglycan modification on mineral formation in a differentiating chick limb-bud mesenchymal cell culture system

In the presence of 4 mM inorganic phosphate, differentiating chick limb-bud mesenchymal cells plated in micromass cultures form a mineralized matrix resembling that of chick calcified cartilage. To test the hypothesis that cartilage proteoglycans are inhibitors of cell mediated mineralization, the synthesis, content, and turnover of proteoglycans were altered in this system, and the extent of mineralization and properties of the mineral crystals examined. In all cases where the proteoglycan synthesis or proteoglycans present were modified to provide fewer or smaller molecules, mineralization was enhanced. Specifically, when proteoglycan synthesis was blocked by treatment with 10⁻¹⁰ M retinoic acid, extensive mineral deposition occurred on a matrix devoid of both proteoglycans and cartilage nodules. The crystals, which formed rapidly, were relatively large in size based on analysis by X-ray diffraction or FT-IR microspectroscopy, and were more abundant than in controls. When 2.5 or 5 mM xylosides were used to cause the synthesis of smaller proteoglycans, the extent of mineral accretion was also increased relative to controls; however, the matrix was less affected, and the extent of mineral deposition and the size of the crystals were not as markedly altered as in the case of retinoic acid. Modification of existing proteoglycans by either chondroinase ABC or hyaluronidase treatment similarly resulted in increased mineral accretion (based on ⁴⁵Ca uptake or total Ca uptake) relative to cultures in which the proteoglycan content was not manipulated. Crystals were more abundant and larger than in control mineralizing cultures. In contrast, when proteoglycan degradation by metalloproteases was inhibited by metal chelation with o-phenanthroline, the Ca accretion at early time points was increased, but as mineralization progressed, Ca accumulation decreased. These data provide evidence that in this culture system, proteoglycans are inhibitors of mineralization. J. Cell. Biochem. 64:632–643. © 1997 Wiley-Liss, Inc.     

INCOMPLETENESS OF THE COCCOSPHERE AS A POSSIBLE STIMULUS FOR COCCOLITH FORMATION IN PLEUROCHRYSIS CARTERAE (PRYMNESIOPHYCEAE)1

Pleurochrysis carterae is a marine biflagellate that produces calcified structures called coccoliths. The coccoliths are formed inside the cells and released from the latter after formation. The light dependence of calcium incorporation in this species was studied using ⁴⁵Ca as a tracer. Cells exposed to a repeating cycle of 16 h of light and 8 h of darkness incorporated calcium in extracellular coccoliths at a more or less constant rate throughout a cycle. The cells divided during the dark periods with a concomitant decrease in size. Their size increased during the light periods. Coccolith formation in cells incubated in continuous darkness was greatly reduced and finally ceased. These cells did not divide and did not increase in size. Removal of extracellular coccoliths prior to the calcium incorporation experiments stimulated coccolith formation both in dark-incubated cells and in cells exposed to a repeating light-dark cycle. Cells in the stationary phase of growth ceased producing coccoliths. Calcification could be induced in these cells by removal of the extracellular coccoliths. Based on these findings we suggest that cells of Pleurochrysis carterae tend to produce a complete cover of coccoliths and that the available cell surface is a factor controlling coccolith formation.     

INCOMPLETENESS OF THE COCCOSPHERE AS A POSSIBLE STIMULUS FOR COCCOLITH FORMATION IN PLEUROCHRYSIS CARTERAE(PRYMNESIOPHYCEAE)1

Pleurochrysis carterae is a marine biflagellate that produces calcified structures called coccoliths. The coccoliths are formed inside the cells and released from the latter after formation. The light dependence of calcium incorporation in this species was studied using⁴⁵Ca as a tracer. Cells exposed to a repeating cycle of 16 h of light and 8 h of darkness incorporated calcium in extracellular coccoliths at a more or less constant rate throughout a cycle. The cells divided during the dark periods with a concomitant decrease in size. Their size increased during the light periods Coccolith formation in cells incubated in continuous darkness was greatly reduced and finally ceased. These cells did not divide and did not increase in size. Removal of extracellular coccoliths prior to the calcium incorporation experiments stimulated coccolith formation both in dark-incubated cells and in cells exposed to a repeating light-dark cycle. Cells in the stationary phase of growth ceased producing coccoliths. Calcification could be induced in these cells by removal of the extracellular coccoliths. Based on these findings we suggest that cells of Pleurochrysis carterae tend to produce a complete cover of coccoliths and that the available cell surface is a factor controlling coccolith formation.     

Morphological and chemical characterization of mineral concretions in the freshwater bivalve Anodonta cygnea (Unionidae)

The freshwater mussel Anodonta cygnea is commonly used as a model organism for biomineralization studies, its peculiar morphofunctional properties also make it an excellent environmental biomonitor. The first detailed on the calcareous concretions from gill and mantle tissue, as well as fluids of the freshwater bivalve A. cygnea, supported by histological, scanning, spectrometry, and spectroscopy analyses. Through these analyses, the morphology, structure, and chemical characterization of these biomineral concretions were accomplished. The concretions represent a high percentage of the dry weight of these organisms. In gill tissue, it can reach up to 50% of dry weight prior to reproductive maturity. Analysis of elemental composition of the tissue concretions showed the presence of calcium and phosphate, as main components, associated with other residual elements like iron, manganese, magnesium, and zinc. Concretions are arranged in concentric alternated layers of organic and inorganic matrix. The shape and size of the concretions vary substantially, from very small, less than 1 μm diameter with very regular round structure, found mainly in the mantle tissue, to more than 50 μm length with irregular globular clusters, found predominantly in the gills. The microstructural organization is of a hydroxyapatite polymorphism in the mantle, in contrast to the gills, which exhibit irregular structure and carbonated hydroxyapatite polymorphism. These differences are supported by higher contents of dinitrogen pentoxide, magnesium, and iron in the mantle concretions, but higher contents of manganese and zinc in the gills. Furthermore, the results indicate that the mineral concretion formation in A. cygnea is a hemocytes reaction to particle or toxic invasions. A second relevant role, concerns the close involvement of these microspherules on the adult and larval shell calcification. J. Morphol. 276:65–76, 2015. © 2014 Wiley Periodicals, Inc.     

Ca2+-dependent ATPase associated with plasma membrane from a calcareous alga, Serraticardia maxima (Corallinaceae, Rhodophyta)

The plasma membrane was isolated from a calcareous red alga, Serraticardia maxima (Yendo) Silva (Corallinaceae), by aqueous two-phase partitioning. Its purity was examined with marker enzymes, Mg²⁺-dependent ATPase, inosine diphosphatase, cytochrome c oxidase and NADH-cytochrome c reductase, as well as the sensitivity of Mg²⁺-dependent ATPase to vanadate, azide and nitrate. The results showed that the isolated plasma membrane was purified enough to study its functions. Electron microscopic observations on thin tissue sections revealed that most vesicles of the isolated plasma membrane were stained by the plasma membrane specific stain, phosphotungstic acid-chromic acid. Mg²⁺- or Ca²⁺-dependent ATPases were associated with the plasma membrane. Ca²⁺-dependent ATPase was activated at physiological cytoplasmic concentrations of Ca²⁺ (0.1–10 μmol/L). However, calmodulin (0.5 μmol/L) did not affect its activity. The pH optimum was 8.0, in contrast to 7.0 for Mg²⁺-dependent ATPase. The isolated plasma membrane vesicles were mostly right side-out. To test for H⁺-translocation, right side-out vesicles were inverted; 27% of vesicles were inside-out after treatment with Triton X-100. The inside-out plasma membrane vesicles showed reduction of quinacrine fluorescence in the presence of 1 mmol/L ATP and 100 μmol/L Ca²⁺. The reduced fluorescence was recovered with the addition of 10 mmol/L NH₄Cl, or 5 μmol/L nigericin plus 50 mmol/L KCl. UTP and CTP substituted for ATP, but ADP did not. Ca²⁺-dependent ATPase might pump H⁺ out in the physiological state. The acidification by this pump might be coupled with alkalinization at the calcifying sites, which induces calcification.     

In vitro formation of mineralized cartilagenous tissue by articular chondrocytes

Summary Study of the deep articular cartilage and adjacent calcified cartilage has been limited by the lack of an in vitro culture system which mimics this region of the cartilage. In this paper we describe a method to generate mineralized cartilagenous tissue in culture using chondrocytes obtained from the deep zone of bovine articular cartilage. The cells were plated on Millipore CM^R filters. The chondrocytes in culture accumulated extracellular matrix and formed cartilagenous tissue which calcified when β-glycerophosphate was added to the culture medium. The cartilagenous tissue generated in vitro contains both type II and type X collagens, large sulfated proteoglycans, and alkaline phosphatase activity. Ultrastructurally, matrix vesicles were seen in the extracellular matrix. Selected area electron diffraction confirmed that the calcification was composed of hydroxyapatite crystals. The chondrocytes, as characterized thus far, appear to maintain their phenotype under these culture conditions which suggests that these cultures could be used as a model to examine the metabolism of cells from the deep zone of cartilage and mineralization of cartilagenous tissue in culture.     

CO2 concentrations in perennially ice-covered lakes of Taylor Valley, Antarctica

Lakes in Taylor Valley, southern Victoria Land,Antarctica, are unusual in that they areperennially covered by a 3–5 m thick ice layer.Previous work on gas concentrations in theselakes has shown that the surface waters aresupersaturated with respect to O₂,N₂O, as well as the noble gases. Our datashow that the dissolved CO₂ (CO_2(aq))concentrations, calculated from pH andCO₂, can be highly undersaturatedat shallow depths of the lakes. CO₂partial pressure values (pCO₂) are as lowas 10^–4.3 atm and 10^–4.2 atm in theeast and west lobes of Lake Bonney,respectively, and 10^–3.8 atm in LakeHoare. CO_2(aq) depletion occurred only inthe uppermost part of the water column, inassociation with elevated primary productivity(PPR). The upward diffusion of CO_2(aq)from the aphotic zone, and the annual input ofCO₂ via glacial meltwater can notreplenish the amount of CO_2(aq) annuallylost to primary productivity in the uppermostmeters of the water column. Calcification is alimited source of CO_2(aq), since the lakesare undersaturated with respect to calcitethrough portions of the austral summer.Preliminary respiration rates have been used toobtain an annual inorganic carbon balance.Further down in the water column, at the sitesof the deep-water maximum in primary production(PPR_max), which in Lakes Bonney andFryxell is associated with nutrient gradients,CO_2(aq) is not undersaturated. A largeupward flux from CO₂-supersaturatedaphotic waters provides a surplus ofCO_2(aq) at the PPR_max. Lake Fryxell,unlike the other lakes, is supersaturated withCO_2(aq) throughout the entire water column.     

Tube construction in the watering pot shell Brechites vaginiferus (Bivalvia; Anomalodesmata; Clavagelloidea)

The bizarre watering pot shells of the clavagellid bivalve Brechites comprise a calcareous tube encrusted frequently with sand grains and other debris, the anterior end of which terminates in a convex perforated plate (the ‘watering pot’). It has not proved easy to understand how such extreme morphologies are produced. Previously published models have proposed that the tube and ‘watering pot’ are formed separately, outside the periostracum, and fuse later. Here we present the results of a detailed study of the structure and repair of the tubes of Brechites vaginiferus which suggest that these models are not correct. Critical observations include the fact that the external surface of the tube and ‘watering pot’ are covered by a thin organic film, on to the inner surface of which the highly organized aragonite crystals are secreted. There is no evidence of a suture between the tube and the ‘watering pot’ or that the periostracum of the juvenile shell passes through the wall of the tube. Live individuals of B. vaginiferus are able to repair substantial holes in the tube or ‘watering pot’ by laying down a new organic film followed by subsequent calcareous layers. Brechites vaginiferus displays Type C mantle fusion, with the result that the whole animal is encased by a continuous ring of mantle and periostracum, thereby making it possible to secrete a continuous ‘ring’ of shell material. On the basis of these observations we suggest that watering pot shells are not extra‐periostracal but are the product of simple modification of ‘normal’ shell‐secreting mechanisms.     

二甲双胍对华法令诱导大鼠动脉钙化的影响及其机制初探

目的:研究二甲双胍(metformin,MET)对华法令(Warfarin,WFN)诱导大鼠动脉钙化的影响及其机制。方法:将28只SD大鼠随机分为正常对照组、8周(W)钙化组、8W钙化+8W MET 100 mg/kg治疗组、8W钙化+8W MET 200 mg/kg治疗组。采用Von Kossa染色法检测胸主动脉组织中钙结节;邻甲酚肽络合酮比色法测定颈总动脉组织中钙沉积含量;免疫组化染色检测血管壁中成骨基因Runx2及血管平滑肌标志物α-SMA的表达;Western Blot检测血管壁中Runx2及自噬标志物LC3II的表达。结果:WFN干预8 W后,大鼠动脉中钙沉积含量显著增加(P0.01),MET治疗组主动脉钙含量与钙化组相比均显著降低(P0.01)。Von Kossa染色可见钙化组(WFN组)动脉壁中层黑色连续钙盐沉积条带,而进行MET治疗后,黑色条带明显减少,对照组未见黑色条带。免疫组化显示钙化组血管壁Runx2表达阳性,棕褐色染色较深,而α-SMA表达则显著下降,基本未见棕褐色染色沉积;MET治疗后能够逆转上述趋势。Western Blot显示钙化组血管壁Runx2表达明显上升,MET治疗后Runx2表达被抑制。此外,钙化过程中伴随着自噬标志物LC3II表达轻度上升;随着MET浓度升高,血管壁中自噬水平呈剂量依赖性显著升高。结论:二甲双胍能够有效抑制大鼠动脉钙化,减轻血管平滑肌细胞由收缩表型向成骨样表型转换,其机制可能与诱导自噬有关。     

Coral reef calcifiers buffer their response to ocean acidification using both bicarbonate and carbonate

Central to evaluating the effects of ocean acidification (OA) on coral reefs is understanding how calcification is affected by the dissolution of CO₂ in sea water, which causes declines in carbonate ion concentration [CO₃²⁻] and increases in bicarbonate ion concentration [HCO₃⁻]. To address this topic, we manipulated [CO₃²⁻] and [HCO₃⁻] to test the effects on calcification of the coral Porites rus and the alga Hydrolithon onkodes, measured from the start to the end of a 15-day incubation, as well as in the day and night. [CO₃²⁻] played a significant role in light and dark calcification of P. rus, whereas [HCO₃⁻] mainly affected calcification in the light. Both [CO₃²⁻] and [HCO₃⁻] had a significant effect on the calcification of H. onkodes, but the strongest relationship was found with [CO₃²⁻]. Our results show that the negative effect of declining [CO₃²⁻] on the calcification of corals and algae can be partly mitigated by the use of HCO₃⁻ for calcification and perhaps photosynthesis. These results add empirical support to two conceptual models that can form a template for further research to account for the calcification response of corals and crustose coralline algae to OA.     

Effects of diurnally oscillating pCO2 on the calcification and survival of coral recruits

Manipulative studies have demonstrated that ocean acidification (OA) is a threat to coral reefs, yet no experiments have employed diurnal variations in pCO(2) that are ecologically relevant to many shallow reefs. Two experiments were conducted to test the response of coral recruits (less than 6 days old) to diurnally oscillating pCO(2); one exposing recruits for 3 days to ambient (440 μatm), high (663 μatm) and diurnally oscillating pCO(2) on a natural phase (420-596 μatm), and another exposing recruits for 6 days to ambient (456 μatm), high (837 μatm) and diurnally oscillating pCO(2) on either a natural or a reverse phase (448-845 μatm). In experiment I, recruits exposed to natural-phased diurnally oscillating pCO(2) grew 6-19% larger than those in ambient or high pCO(2). In experiment II, recruits in both high and natural-phased diurnally oscillating pCO(2) grew 16 per cent larger than those at ambient pCO(2), and this was accompanied by 13-18% higher survivorship; the stimulatory effect on growth of oscillatory pCO(2) was diminished by administering high pCO(2) during the day (i.e. reverse-phased). These results demonstrate that coral recruits can benefit from ecologically relevant fluctuations in pCO(2) and we hypothesize that the mechanism underlying this response is highly pCO(2)-mediated, night-time storage of dissolved inorganic carbon that fuels daytime calcification.     

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.     

Human-derived nanoparticles and vascular response to injury in rabbit carotid arteries: proof of principle

Self-calcifying, self-replicating nanoparticles have been isolated from calcified human tissues. However, it is unclear if these nanoparticles participate in disease processes. Therefore, this study was designed to preliminarily test the hypothesis that human-derived nanoparticles are causal to arterial disease processes. One carotid artery of 3 kg male rabbits was denuded of endothelium; the contralateral artery remained unoperated as a control. Each rabbit was injected intravenously with either saline, calcified, or decalcified nanoparticles cultured from calcified human arteries or kidney stones. After 35 days, both injured and control arteries were removed for histological examination. Injured arteries from rabbits injected with saline showed minimal, eccentric intimal hyperplasia. Injured arteries from rabbits injected with calcified kidney stone- and arterial-derived nanoparticles occluded, sometimes with canalization. The calcified kidney stone-derived nanoparticles caused calcifications within the occlusion. Responses to injury in rabbits injected with decalcified kidney stone-derived nanoparticles were similar to those observed in saline-injected animals. However, decalcified arterial-derived nanoparticles produced intimal hyperplasia that varied from moderate to occlusion with canalization and calcification. This study offers the first evidence that there may be a causal relationship between human-derived nanoparticles and response to injury including calcification in arteries with damaged endothelium.