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Patients with chronic kidney disease (CKD) have a higher incidence of vascular calcification and a greatly increased risk of cardiovascular death. The mechanisms involved in the accelerated vascular calcification observed in CKD have recently become clearer, leading to the hypothesis that a lack of natural inhibitors of calcification may trigger calcium deposition. One of these inhibitory factors, matrix Gla protein (MGP), is the focus of the present review. MGP, originally isolated from bone, is a vitamin K-dependent protein that is also highly expressed by vascular smooth muscle cells. MGP has been confirmed as a calcification-inhibitor in numerous studies; however, its mechanism of action is not completely understood. It potentially acts in several ways to regulate calcium deposition including: (i) binding calcium ions and crystals; (ii) antagonizing bone morphogenetic protein and altering cell differentiation; (iii) binding to extracellular matrix components; and (iv) regulating apoptosis. Its expression is regulated by several factors including retinoic acid, vitamin D and extracellular calcium ions, and a reduced form of vitamin K (KH2) is important in maintaining MGP in an active form. Therefore, strategies aimed at increasing its expression and activity may be beneficial in tipping the balance in favour of inhibition of calcification in CKD.

The nuclear envelope defines the barrier between the nucleus and cytoplasm and features inner and outer membranes separated by a perinuclear space (PNS). The inner nuclear membrane contains specific integral proteins that include Sun1 and Sun2. Although the outer nuclear membrane (ONM) is continuous with the endoplasmic reticulum, it is nevertheless enriched in several integral membrane proteins, including nesprin 2 Giant (nesp2G), an 800-kD protein featuring an NH2-terminal actin-binding domain. A recent study (Padmakumar, V.C., T. Libotte, W. Lu, H. Zaim, S. Abraham, A.A. Noegel, J. Gotzmann, R. Foisner, and I. Karakesisoglou. 2005. J. Cell Sci. 118:3419–3430) has shown that localization of nesp2G to the ONM is dependent upon an interaction with Sun1. In this study, we confirm and extend these results by demonstrating that both Sun1 and Sun2 contribute to nesp2G localization. Codepletion of both of these proteins in HeLa cells leads to the loss of ONM-associated nesp2G, as does overexpression of the Sun1 lumenal domain. Both treatments result in the expansion of the PNS. These data, together with those of Padmakumar et al. (2005), support a model in which Sun proteins tether nesprins in the ONM via interactions spanning the PNS. In this way, Sun proteins and nesprins form a complex that links the nucleoskeleton and cytoskeleton (the LINC complex).

The high rates of atherosclerotic vascular disease in patients with end-stage renal disease (ESRD) cannot be fully explained by the excess of traditional risk factors. Interest has therefore arisen in the possible role of vascular calcification, which is increased in these patients and may effect plaque stability and have detrimental hemodynamic consequences. Considerable evidence has accumulated recently pointing to the regulated nature of the calcification process. The initiation of calcium crystal formation appears to require the presence of small membrane bound vesicles released by living or apoptotic cells. The cellular release, content and phagocytosis of these vesicles appear to be important regulatory pathways in vascular calcification. Better understanding of these mechanisms may have therapeutic potential in reducing the adverse cardiovascular event rates in patients with (ESRD).

Nesprin-2 is a multi-isomeric, modular protein composed of variable numbers of spectrin-repeats linked to a C-terminal transmembrane domain and/or to N-terminal paired calponin homology (CH) domains. The smaller isoforms of nesprin-2 co-localize with and bind lamin A and emerin at the inner nuclear envelope (NE). In SW-13 cells, which lack lamin A/C, nesprin-2 epitopes and emerin were both mislocalized and formed aggregates in the endoplasmic reticulum (ER). The larger isoforms and other CH-domain-containing isoforms co-localize with heterochromatin within the nucleus and are also present at the outer NE and in multiple cytoplasmic compartments. Nesprin-2 isoforms relocalize during in vitro muscle differentiation of C2C12 myoblasts to the sarcomere of myotubes. Immunogold electron microscopy using antibodies specific for three different epitopes detected nesprin-2 isoforms at multiple locations including intranuclear foci, both membranes of the NE, mitochondria, sarcomeric structures and plasma membrane foci. In adult skeletal muscle, confocal immunolocalization studies demonstrated that nesprin-2 epitopes were present at the Z-line and were also associated with the sarcoplasmic reticulum (SR) in close apposition to SERCA2. These data suggest that nesprin-2 isoforms form a linking network between organelles and the actin cytoskeleton and thus may be important for maintaining sub-cellular spatial organisation. Moreover, its association at the NE with lamin and emerin, the genes mutated in Emery-Dreifuss muscular dystrophy, suggests a mechanism to explain how disruption of the NE leads to muscle dysfunction.

Vascular calcification is commonplace in patients with end-stage renal disease where it develops rapidly and predicts a variety of adverse outcomes. The processes responsible for vascular calcification have been the focus of much research, aided in recent decades by molecular genetic techniques and in vitro models. Converging evidence now suggests that vascular calcification is an active, regulated process, with abundant similarities to the process of skeletal mineralization. Using an in vitro model of calcifying vascular smooth muscle cells (VSMCs), we have shown that a mineral imbalance induces VSMC apoptosis, and that VSMC apoptotic bodies and vesicles can nucleate basic calcium phosphate in the form of hydroxyapatite, the same mineral found in bone. Gene expression studies suggest that the normal vessel wall expresses proteins such as matrix Gla protein that inhibit calcification. In addition, circulating proteins such as fetuin-A are produced at remote sites and act to inhibit soft tissue calcification systemically. However, down-regulation or perturbation of these proteins may lead to a phenotypic transformation of VSMCs to osteo/chondrocytic-like cells while the calcified environment may stimulate macrophages to adopt osteoclastic properties. Both clinical and basic research findings indicate an inverse relationship between bone mineralization and vascular calcification. The mechanisms linking these two processes are a topic for further investigation, with current theories proposing a role for lipids, common regulatory molecules, and calcium and bone turnover. We have synthesized these findings into a theoretical model offering a putative pathway for the development of severe vascular calcification in end-stage renal disease.

Vascular calcification is an actively regulated process driven by vascular smooth muscle cell (VSMC) adaptation and ultimately dysfunction, leading to the induction of active osteogenic processes within the vessel wall. Dai et al., for the first time, identify autophagy as a novel adaptive mechanism that protects against phosphate-induced VSMC calcification, by acting to regulate apoptosis and the release of mineralizing matrix vesicles from VSMCs.

Implants of collagen-fibronectin gels containing Bcl-2-transduced human umbilical vein endothelial cells (Bcl-2-HUVECs) induce the formation of human endothelial cell (EC)/murine vascular smooth muscle cell (VSMC) chimeric vessels in immunodeficient mice. Microfil casting of the vasculature 60 d after implantation reveals highly branched microvascular networks within the implants that connect with and induce remodeling of conduit vessels arising from the abdominal wall circulation. Approximately 85% of vessels within the implants are lined by Bcl-2-positive human ECs expressing VEGFR1, VEGFR2, and Tie-2, but not integrin αvβ3. The human ECs are seated on a well formed human laminin/collagen IV-positive basement membrane, and are surrounded by mouse VSMCs expressing SM-α actin, SM myosin, SM22α, and calponin, all markers of contractile function. Transmission electron microscopy identified well formed EC–EC junctions, chimeric arterioles with concentric layers of contractile VSMC, chimeric capillaries surrounded by pericytes, and chimeric venules. Bcl-2-HUVEC-lined vessels retain 70-kDa FITC-dextran, but not 3-kDa dextran; local histamine rapidly induces leak of 70-kDa FITC-dextran or India ink. As in skin, TNF induces E-selectin and vascular cell adhesion molecule 1 only on venular ECs, whereas intercellular adhesion molecule-1 is up-regulated on all human ECs. Bcl-2-HUVEC implants are able to engraft within and increase perfusion of ischemic mouse gastrocnemius muscle after femoral artery ligation. These studies show that cultured Bcl-2-HUVECs can differentiate into arterial, venular, and capillary-like ECs when implantedin vivo, and induce arteriogenic remodeling of the local mouse vessels. Our results support the utility of differentiated EC transplantation to treat tissue ischemia.

Vascular smooth muscle cell (VSMC) differentiation and phenotypic modulation is characterized by changes in mRNA expression for smooth muscle (SM) marker contractile proteins such as alpha-SM actin and SM22 alpha. Transforming growth factor beta1 (TGF-beta 1) is a potent VSMC differentiation factor; however, it is not known if other TGF-beta-superfamily members, in particular the bone morphogenetic proteins (BMPs), modulate VSMC phenotype. Here we demonstrate that a large subset of TGF-beta-superfamily members and their type I receptors are differentially co-expressed as VSMC phenotype changes during fetal/neonatal development and that BMP2, -4, and -6 reciprocally regulate SM-marker mRNA and protein expression in vitro. BMP2 and BMP6 decrease expression of the SM markers alpha-SM actin, SM22alpha, and calponin in rat VSMCs, whereas BMP4 increases their expression. The effects of BMP-2, -4, and -6 on SM marker gene transcription are mediated through a consensus TGF-beta-controlling element, the TCE, which is common to regulatory regions of SM-marker genes. Moreover, co-treatment experiments revealed that BMP-2, -4, and -6 each inhibit TGF-beta 1-modulated increases in SM22alpha reporter gene activity. Regardless of whether they positively or negatively regulate SM marker expression, TGF-beta 1 and BMP-2, -4, and -6 all induced binding of the Krüppel-like transcription factor, GKLF/KLF4, to the TGF-beta control element. Induction of KLF4 was confirmed by immunocytochemistry and Western blotting, which revealed that a lower molecular weight KLF4 protein is induced after treatment with TGF-beta-superfamily members. Taken together, our results demonstrate that multiple members of the TGF-beta superfamily act in concert to modulate VSMC phenotype.

Objective— Mineralization-regulating proteins are found deposited at sites of vascular calcification. However, the relationship between the onset of calcification in vivo and the expression of genes encoding mineralization-regulating proteins is unknown. This study aimed to determine the temporal and spatial pattern of expression of key bone and cartilage proteins as atherosclerotic calcification progresses. Methods and Results— Using reverse transcription-polymerase chain reaction on a panel of noncalcified and calcified human arterial samples, two classes of proteins could be identified: (1) Matrix Gla protein, osteonectin, osteoprotegerin, and aggrecan were constitutively expressed by vascular smooth muscle cells (VSMCs) in the normal vessel media but downregulated in calcified arteries whereas (2) alkaline phosphatase, bone sialoprotein, osteocalcin, and collagen II were expressed predominantly in the calcified vessel together with Cbfa1, Msx2, and Sox9, transcription factors that regulate expression of these genes. In the calcified plaque in situ hybridization identified subsets of VSMCs expressing osteoblast and chondrocyte-like gene expression profiles whereas osteoclast-like macrophages were present around sites of calcification. Conclusions— These observations suggest a sequence of molecular events in vascular calcification beginning with the loss of expression by VSMCs, of constitutive inhibitory proteins, and ending with expression by VSMCs and macrophages of chondrocytic, osteoblastic, and osteoclastic-associated proteins that orchestrate the calcification process.

Background— Vascular smooth muscle cells (VSMCs) in atherosclerotic lesions display an osteogenic phenotype, and calcification commonly occurs in association with lipid. We therefore tested the hypothesis that lipid components in atherosclerotic lesions influenced VSMC phenotype and calcification using an in vitro model of calcification. Methods and Results— In situ hybridization of human atherosclerotic plaques (n=10) collected from patients undergoing carotid endarterectomy demonstrated that subsets of lipid-filled VSMCs adjacent to sites of calcification expressed alkaline phosphatase, bone Gla protein, and bone sialoprotein, suggesting an osteogenic phenotype. Treatment of VSMCs in culture with acetylated low-density lipoprotein (acLDL) or lipoprotein-deficient serum altered the time course of bone-associated protein gene expression and calcification. AcLDL increased nodule calcification 3-fold, whereas lipoprotein-deficient serum significantly inhibited it. Reverse transcriptase–polymerase chain reaction and Western analysis demonstrated the presence of the acLDL receptor, SRA1, exclusively in calcifying nodular VSMCs, and blockade of SRA with polyinosinic acid inhibited acLDL-induced calcification. Because apoptotic bodies can serve as nucleation sites for calcification, we investigated whether acLDL could stimulate apoptosis in nodules. Apoptosis of nodular VSMCs was unaltered, but the number of apoptotic bodies per nodule increased ≈3-fold, implying a defect in phagocytosis. Consistent with these observations, binding of apoptotic bodies to VSMCs was decreased in the presence of acLDL. Conclusions— These studies suggest that modified lipoproteins stimulate calcification by enhancing osteogenic differentiation of VSMCs and by a novel mechanism whereby acLDL interacts with SRA on VSMCs and blocks phagocytic removal of apoptotic bodies.

Nesprin-1 and nesprin-2 (also known as Syne-1 and Syne-2,) are large ( approximately 3300-residue) vertebrate proteins associated with emerin and lamin A at the nuclear envelope of muscle cells and other cell types. We show that the previously described nesprins are short isoforms of giant proteins comprising an actin-binding amino-terminus connected to a carboxy-terminal klarsicht-related transmembrane domain by a massive ( approximately 6000-8000 amino acid) spectrin-like rod domain, making full-length nesprin-1, at one megadalton, the largest non-titin protein hitherto described in humans. We find that MSP-300, a 7000-residue Drosophila melanogaster protein whose disruption results in defects of muscle development, corresponds to the N-terminal two-thirds of the Drosophila nesprin ortholog. A nesprin-like protein is also encoded by the nematode genome. Moreover, we demonstrate that the larger isoforms of nesprin-1, like MSP-300, are localized to the sarcomeric Z-line of both skeletal and cardiac muscle. The recognition that a characteristic muscle-specific mutant phenotype in the fly results from a disruption of its nesprin ortholog reinforces the candidacy of the human proteins for involvement in genetic diseases of skeletal and cardiac muscle.

YAP is a mechanosensitive transcriptional activator with a critical role in cancer, regeneration, and organ size control. Here, we show that force applied to the nucleus directly drives YAP nuclear translocation by decreasing the mechanical restriction of nuclear pores to molecular transport. Exposure to a stiff environment leads cells to establish a mechanical connection between the nucleus and the cytoskeleton, allowing forces exerted through focal adhesions to reach the nucleus. Force transmission then leads to nuclear flattening, which stretches nuclear pores, reduces their mechanical resistance to molecular transport, and increases YAP nuclear import. The restriction to transport is further regulated by the mechanical stability of the transported protein, which determines both active nuclear transport of YAP and passive transport of small proteins. Our results unveil a mechanosensing mechanism mediated directly by nuclear pores, demonstrated for YAP but with potential general applicability in transcriptional regulation.

Introduction Vascular calcification is a hallmark of vascular ageing, and associated with vascular smooth muscle cell (VSMC) death, phenotype modulation and maladaptation. However, it remains unclear how the initial stress signals link to these downstream cellular events. Emerging evidence and our in vitro data suggest that stress may drive vascular calcification through elevated levels of DNA damage, a key factor driving cellular ageing. Methods and Results We investigated the effects of different DNA damage signalling pathway inhibitors (ATM, ATM/ATR and PARP1) on the progression of vascular calcification. Using comet assays and western blot, we found elevated levels of DNA damage in calcified VSMCs and that inducing DNA damage accelerated rates of calcification. Chemical inhibition or siRNA knockdown of ATM, ATM/ATR or PARP signalling reduced or delayed calcification and prevented cells undergoing calcification-associated phenotype changes including osteo/chondrogenic differentiation. Prevention was associated with down-regulation of senescence and inflammatory markers suggesting the senescence associated secretory phenotype (SASP) acts to potentiate VSMC calcification. Conclusion Taken together, these in vitro data suggest DNA damage signalling is involved in the pathological regulation of calcification. Therefore, interventions that reduce DNA damage, promote DNA damage repair, or modulate DNA damage signalling could be potential therapies for the prevention of vascular calcification.

Osteocalcin, or bone Gla protein, is a small protein secreted by osteoblasts that can undergo γ-carboxylation. The γ-carboxylated form binds hydroxyapatite and is abundant in bone extracellular matrix. In contrast, the undercarboxylated circulating form has been implicated as a novel hormone and positive regulator of glucose homeostasis. Importantly, osteocalcin expression has been described in calcifying vascular smooth muscle cells (VSMCs), although the physiological significance of this observation has remained unclear. In this issue of Arteriosclerosis, Thrombosis, and Vascular Biology , Idelevich et al1 show that osteocalcin is potentially a novel regulator of osteochondrogenic differentiation of pathologically mineralizing VSMCs. They reveal that osteocalcin, via hypoxia-inducible factor 1α signaling, stimulates expression of osteochondrogenic transcription factors in VSMCs, as well as a shift in cellular metabolism toward glycolysis. This study provides the first evidence that osteocalcin may be an active player in vascular calcification, with its presence in the calcified vasculature, and potentially the circulation, activating novel signaling pathways that promote mineralization. See accompanying article: Idelevich A, Rais Y, Monsonego-Ornan E. Bone Gla protein increases HIF-1α-dependent glucose metabolism and induces cartilage and vascular calcification. Arterioscler Thromb Vasc Biol . 2011;31:e55–e71. Pathological mineralization of the vasculature has a detrimental effect on cardiovascular function and is associated with increased mortality in patients with aging, atherosclerosis, type 2 diabetes, and chronic kidney disease.2 Vascular smooth muscle cells (VSMCs) orchestrate the mineralization process, which is mediated in part by their osteochondrocytic differentiation in the vessel wall. This phenotypic transition is characterized by expression of Runx2 and Sox9, master transcription factors that regulate bone and cartilage differentiation during developmental osteochondrogenesis, as well as other bone- and cartilage-specific proteins, many with undefined functions.3 One of these proteins, osteocalcin, is a small γ-carboxylated protein that is expressed by both osteoblasts and VSMCs and abundantly deposited …

Ectopic calcification of the vasculature comes with age and is also prevalent in patients with chronic kidney disease (CKD). Klotho, a protein highly expressed in the kidney, has been identified as a novel anti-aging factor,[1][1],[2][2] and Klotho-deficient animals develop medial vascular

In eukaryotic cells, non-translating mRNAs can accumulate into cytoplasmic mRNP (messenger ribonucleoprotein) granules such as P-bodies (processing bodies) and SGs (stress granules). P-bodies contain the mRNA decay and translational repression machineries and are ubiquitously expressed in mammalian cells and lower eukaryote species including Saccharomyces cerevisiae, Drosophila melanogaster and Caenorhabditis elegans. In contrast, SGs are only detected during cellular stress when translation is inhibited and form from aggregates of stalled pre-initiation complexes. SGs and P-bodies are related to NGs (neuronal granules), which are essential in the localization and control of mRNAs in neurons. Importantly, RNA granules are linked to the cytoskeleton, which plays an important role in mediating many of their dynamic properties. In the present review, we discuss how P-bodies, SGs and NGs are linked to cytoskeletal networks and the importance of these linkages in maintaining localization of their RNA cargoes.

International audience; Vascular calcifications (VCs) are actively regulated biological processes associated with crystallization of hydroxyapatite in the extracellular matrix and in cells of the media (VCm) or intima (VCi) of the arterial wall. Both patterns of VC often coincide and occur in patients with type II diabetes, chronic kidney disease, and other less frequent disorders; VCs are also typical in senile degeneration. In this article, we review the current state of knowledge about the pathology, molecular biology, and nosology of VCm, expand on potential mechanisms responsible for poor prognosis, and expose some of the directions for future research in this area.

Abstract —The mechanisms involved in the initiation of vascular calcification are not known, but matrix vesicles, the nucleation sites for calcium crystal formation in bone, are likely candidates, because similar structures have been found in calcified arteries. The regulation of matrix vesicle production is poorly understood but is thought to be associated with apoptotic cell death. In the present study, we investigated the role of apoptosis in vascular calcification. We report that apoptosis occurs in a human vascular calcification model in which postconfluent vascular smooth muscle cell (VSMC) cultures form nodules spontaneously and calcify after ≈28 days. Apoptosis occurred before the onset of calcification in VSMC nodules and was detected by several methods, including nuclear morphology, the TUNEL technique, and external display of phosphatidyl serine. Inhibition of apoptosis with the caspase inhibitor ZVAD.fmk reduced calcification in nodules by ≈40%, as measured by the cresolphthalein method and alizarin red staining. In addition, when apoptosis was stimulated in nodular cultures with anti-Fas IgM, there was a 10-fold increase in calcification. Furthermore, incubation of VSMC-derived apoptotic bodies with 45 Ca demonstrated that, like matrix vesicles, they can concentrate calcium. These observations provide evidence that apoptosis precedes VSMC calcification and that apoptotic bodies derived from VSMCs may act as nucleating structures for calcium crystal formation.

Matrix Gla protein (MGP) is a mineral binding extra-cellular matrix protein which is thought to be a key inhibitor of tissue and vascular calcification. It is known to be upregulated in areas of extracellular calcification possibly to limit further harmful calcification. In this study we have demonstrated that extracellular ionic calcium (high levels of which induce calcification) is a key signal for MGP regulation and that this effect is mediated by a G protein mediated cation-sensing mechanism, functionally related to, but molecularly distinct from the calcium-sensing receptor. We therefore propose that this novel cation sensing mechanism may play a homeostatic role in preventing pathological calcification.

These studies aim to investigate the expression and function of mineralisation-regulating proteins in association with human vascular calcification focussing on the similarities and differences between the two major calcification pathologies in man: atherosclerotic, intimal calcification and Monckeberg's sclerotic medial calcification.A number of studies have documented expression of mineralisation-regulating proteins in association with human atherosclerotic calcification leading to the suggestion that human vascular calcification may be a regulated process with similarities to developmental osteogenesis.In situ hybridisation, immunohistochemistry and semi-quantitative RT-PCR analysis were used to determine the temporal and spatial expression patterns of mineralisation-regulating proteins within human calcified vascular lesions. Additionally, the expression and regulation of bone-associated proteins was analysed during spontaneous calcification of human VSMCs in vitro.In association with both medial and intimal calcification, the temporal changes in expression of mineralisation-regulating proteins are similar. Some constitutively expressed bone-associated proteins, including matrix Gla protein (MGP), are down-regulated in association with calcification while expression of a number of bone-associated proteins, not normally expressed in the vessel wall, are induced including alkaline phosphatase (ALK), bone sialoprotein (BSP) and bone Gla protein (BGP). In medial calcification the source of expression of these mineralisation-regulating proteins is VSMCs while in intimal lesions both VSMCs and macrophages express them. Furthermore, these bone-associated proteins are spontaneously expressed by VSMCs in vitro suggesting that human VSMCs are capable of simultaneously exhibiting smooth muscle and osteogenic-like properties.These studies imply that both medial and intimal vascular calcification are regulated processes; however the aetiology of each pathology differs.

Background —Calcification of the media of peripheral arteries is referred to as Mönckeberg’s sclerosis (MS) and occurs commonly in aged and diabetic individuals. Its pathogenesis is unknown, but its presence predicts risk of cardiovascular events and leg amputation in diabetic patients. Several studies have documented expression of bone-associated genes in association with intimal atherosclerotic calcification, leading to the suggestion that vascular calcification may be a regulated process with similarities to developmental osteogenesis. Therefore, we examined gene expression in vessels with MS to determine whether there was evidence for a regulated calcification process in the vessel media. Methods and Results —In situ hybridization, immunohistochemistry, and semiquantitative reverse-transcription polymerase chain reaction were used to examine the expression of mineralization-regulating proteins in human peripheral arteries with and without MS. MS occurred in direct apposition to medial vascular smooth muscle cells (VSMCs) in the absence of macrophages or lipid. These VSMCs expressed the smooth muscle–specific gene SM22α and high levels of matrix Gla protein but little osteopontin mRNA. Compared with normal vessels, vessels with MS globally expressed lower levels of matrix Gla protein and osteonectin, whereas alkaline phosphatase, bone sialoprotein, bone Gla protein, and collagen II, all indicators of osteogenesis/chondrogenesis, were upregulated. Furthermore, VSMCs derived from MS lesions exhibited osteoblastic properties and mineralized in vitro. Conclusions —These data indicate that medial calcification in MS lesions is an active process potentially orchestrated by phenotypically modified VSMCs.

Pyrophosphate is a potent inhibitor of medial vascular calcification where its level is controlled by hydrolysis via a tissue-nonspecific alkaline phosphatase (TNAP). We sought to determine if increased TNAP activity could explain the pyrophosphate deficiency and vascular calcification seen in renal failure. TNAP activity increased twofold in intact aortas and in aortic homogenates from rats made uremic by feeding adenine or by 5/6 nephrectomy. Immunoblotting showed an increase in protein abundance but there was no increase in TNAP mRNA assessed by quantitative polymerase chain reaction. Hydrolysis of pyrophosphate by rat aortic rings was inhibited about half by the nonspecific alkaline phosphatase inhibitor levamisole and was reduced about half in aortas from mice lacking TNAP. Hydrolysis was increased in aortic rings from uremic rats and all of this increase was inhibited by levamisole. An increase in TNAP activity and pyrophosphate hydrolysis also occurred when aortic rings from normal rats were incubated with uremic rat plasma. These results suggest that a circulating factor causes pyrophosphate deficiency by regulating TNAP activity and that vascular calcification in renal failure may result from the action of this factor. If proven by future studies, this mechanism will identify alkaline phosphatase as a potential therapeutic target.

Abstract —Early morphological and biochemical studies indicated that vascular smooth muscle cells (VSMCs) exhibited two distinct phenotypes and that a change from the contractile to the synthetic phenotype was a prerequisite for progression of vascular disease. More recently, it has become evident that these phenotypes probably represent the extremes of a spectrum of phenotypes that may coexist in the vessel wall, which are dictated by their environment and functional requirements and which reflect differing patterns of gene expression. Therefore, knowledge of the key factors that regulate these patterns of gene expression is likely to lead to the ability to manipulate VSMC phenotype. However, before such factors can be identified, the relationship between VSMC gene expression and VSMC phenotype must first be established. We therefore undertook a differential screen of cDNA from VSMCs in vitro to provide a bank of gene markers that could be used under a variety of circumstances to define VSMC phenotype in terms of the pattern of genes expressed. Using this approach, we have found that the pattern of gene expression that occurs during neointima formation in the balloon-injured rat carotid artery is very similar to that seen at a specific period in the developing aorta of the early neonate and is characterized by coexpression of genes for both contractile and matrix proteins. Furthermore, recent studies have shown that VSMCs isolated at different stages of aortic development can stably maintain different phenotypic characteristics in cell culture. The use of these cells in transfection experiments with SM-specific promoter–chloramphenicol acetyltransferase reporter constructs may enable us to determine what regulates the pattern of gene expression in different VSMC phenotypes. Such studies may ultimately lead to the identification of transcription factors responsible for determining VSMC phenotype and may therefore provide targets for therapy aimed at manipulating VSMC gene expression in vascular disease.

Abstract —The cellular and molecular events leading to calcification in atherosclerotic lesions are unknown. We and others have shown that bone-associated proteins, particularly matrix Gla protein (MGP) and osteopontin (OP), can be detected in atherosclerotic lesions, thus suggesting an active calcification process. In the present study, we aimed to determine whether human vascular smooth muscle cells (VSMCs) could calcify in vitro and to determine whether MGP and OP have a role in vascular calcification. We established that human aortic VSMCs and placental microvascular pericytes spontaneously form nodules in cell culture and induce calcification, as detected by von Kossa’s method, Alizarin red S staining, and electron microscopy. The cells in calcifying nodules differed from those in monolayer cultures by expressing higher levels of the SMC markers α-SM actin, SM22α, and calponin. In addition, Northern blot analysis revealed that in human VSMCs, calcification was associated with increased levels of MGP mRNA. In contrast, OP mRNA was barely detectable in calcified human VSMCs and pericyte nodules, nor was OP protein detected, suggesting that OP was not necessary for calcification to occur. These studies reveal that human VSMCs are capable of inducing calcification and that MGP may have a role in human vascular calcification.

Nesprins are large multi-domain proteins that link the nuclear envelope to the cytoskeleton and nucleoskeleton. Here we show that nesprin-1 and nesprin-2 play important roles in regulating cell shape and migration in endothelial cells. Nesprin-1 or nesprin-2 depletion by RNAi increased endothelial cell spread area and the length of cellular protrusions, as well as stimulating stress fibre assembly which correlated with an increase in F-actin levels. Nuclear area was also increased by nesprin depletion, and localization of the inner nuclear membrane protein emerin to the nuclear envelope was reduced. Depletion of nesprin-1 or nesprin-2 reduced migration of endothelial cells into a cell-free area, and decreased loop formation in an in vitro angiogenesis assay. Taken together, our results indicate that nesprin-1 and nesprin-2 both regulate nuclear and cytoplasmic architecture, which we propose leads to their effects on endothelial cell migration and angiogenic loop formation.

> ### Circulation Research Compendium on Aortic Valve Disease: > > Introduction to the Compendium > > Introduction to Aortic Stenosis > > Hemodynamic and Cellular Response Feedback in Calcific Aortic Valve Disease > > Molecular and Cellular Aspects of Calcific Aortic Valve Disease > > Fibrocalcific Aortic Valve Disease > > Current Management of Calcific Aortic Stenosis > > Donald Heistad & Linda Demer, Editors Once symptoms arise, stenotic aortic valve disease leads rapidly to death unless surgical or interventional replacement is performed. Already the most prevalent form of valvular heart disease in the Western world, fibrocalcific aortic valve disease affects 25% of those aged >65 years,1 and its incidence is growing inevitably as our population ages. Valve replacement is second only to coronary bypass surgery among operations in the heart. Food and Drug Administration approval of percutaneous transcatheter aortic valve replacement this past year has sparked growing interest in the underlying pathophysiology of the disease. According to many of our textbooks, aortic valve stenosis results from an inevitable, degenerative disorder of aging, due solely to mechanical wear and tear. However, with advances in experimental models and based on pioneering work2–4 by Mohler, Otto, and Rajamannan, recent research findings are now converging on a major shift in paradigm, with inflammation and …

Rationale: Vascular calcification is prevalent in the aging population, yet little is known of the mechanisms driving age-associated vascular smooth muscle cell (VSMC) phenotypic change. Objective: To investigate the role of nuclear lamina disruption, a specific hallmark of VSMC aging, in driving VSMC osteogenic differentiation. Methods and Results: Prelamin A, the unprocessed form of the nuclear lamina protein lamin A, accumulated in calcifying human VSMCs in vitro and in vivo, and its overexpression promoted VSMC osteogenic differentiation and mineralization. During VSMC aging in vitro, prelamin A accumulation occurred concomitantly with increased p16 expression and osteogenic differentiation and was associated with increased levels of DNA damage. Microarray analysis showed that DNA damage repair pathways were significantly impaired in VSMCs expressing prelamin A and that chemical inhibition and siRNA depletion of the DNA damage response kinases ataxia–telangiectasia mutated/ataxia-telangiectasia– and Rad3-related effectively blocked VSMC osteogenic differentiation and mineralization. In coculture experiments, prelamin A–expressing VSMCs induced alkaline phosphatase activity in mesenchymal progenitor cells, and this was abrogated by inhibition of ataxia-telangiectasia–mutated signaling, suggesting that DNA damage induces the secretion of pro-osteogenic factors by VSMCs. Cytokine array analysis identified several ataxia-telangiectasia mutated–dependent senescence-associated secretory phenotype factors/cytokines released by prelamin A–positive VSMCs, including the calcification regulators bone morphogenetic protein 2, osteoprotegerin, and interleukin 6. Conclusions: Prelamin A promotes VSMC calcification and aging by inducing persistent DNA damage signaling, which acts upstream of VSMC osteogenic differentiation and the senescence-associated secretory phenotype. Agents that target the DNA damage response and prelamin A toxicity may be potential therapies for the treatment of vascular calcification.

Rationale: We previously showed that early calcification of atherosclerotic plaques associates with macrophage accumulation. Chronic renal disease and mineral imbalance accelerate calcification and the subsequent release of matrix vesicles (MVs), precursors of microcalcification. Objective: We tested the hypothesis that macrophage-derived MVs contribute directly to microcalcification. Methods and Results: Macrophages associated with regions of calcified vesicular structures in human carotid plaques (n=136 patients). In vitro, macrophages released MVs with high calcification and aggregation potential. MVs expressed exosomal markers (CD9 and TSG101) and contained S100A9 and annexin V. Silencing S100A9 in vitro and genetic deficiency in S100A9 −/− mice reduced MV calcification, whereas stimulation with S100A9 increased calcification potential. Externalization of phosphatidylserine after Ca/P stimulation and interaction of S100A9 and annexin V indicated that a phosphatidylserine-annexin V-S100A9 membrane complex facilitates hydroxyapatite nucleation within the macrophage-derived MV membrane. Conclusions: Our results support the novel concept that macrophages release calcifying MVs enriched in S100A9 and annexin V, which contribute to accelerated microcalcification in chronic renal disease.

There is continuing debate about whether abundant citrate plays an active role in biomineralization of bone. Using solid state NMR dipolar dephasing, we examined another normally mineralized hard tissue, mineralized articular cartilage, as well as biocalcifications arising in pathological conditions, mineralized intimal atherosclerotic vascular plaque, and apatitic uroliths (urinary stones). Residual nondephasing ¹³C NMR signal at 76 ppm in the spectra of mineralized cartilage and vascular plaque indicates that a quaternary carbon atom resonates at this frequency, consistent with the presence of citrate. The presence, and as yet unproven possible mechanistic involvement, of citrate in tissue mineralization extends the compositional, structural, biogenetic, and cytological similarities between these tissues and bone itself. Out of 10 apatitic kidney stones, five contained NMR-detectable citrate. Finding citrate in a high proportion of uroliths may be significant in view of the use of citrate in urolithiasis therapy and prophylaxis. Citrate may be essential for normal biomineralization (e.g., of cartilage), play a modulatory role in vascular calcification which could be a target for therapeutic intervention, and drive the formation of apatitic rather than other calcific uroliths, including more therapeutically intractable forms of calcium phosphate.

Nuclearenvelopespectrin-repeatproteins(Nesprins), are a novel family of nuclear and cytoskeletal proteins with rapidly expanding roles as intracellular scaffolds and linkers. Originally described as proteins that localise to the nuclear envelope (NE) and establish nuclear-cytoskeletal connections, nesprins have now been found to comprise a diverse spectrum of tissue specific isoforms that localise to multiple sub-cellular compartments. Here, we describe how nesprins are necessary in maintaining cellular architecture by acting as essential scaffolds and linkers at both the NE and other sub-cellular domains. More importantly, we speculate how nesprin mutations may disrupt tissue specific nesprin scaffolds and explain the tissue specific nature of many nesprin-associated diseases, including laminopathies.

Cardiovascular disease begins early in the course of renal decline and is a life-limiting problem in patients with CKD. The increased burden of cardiovascular disease is due, at least in part, to calcification of the vessel wall. The uremic milieu provides a perfect storm of risk factors for accelerated calcification, but elevated calcium and phosphate levels remain key to the initiation and progression of vascular smooth muscle cell calcification in CKD. Vascular calcification is a highly regulated process that involves a complex interplay between promoters and inhibitors of calcification and has many similarities to bone ossification. Here, we discuss current understanding of the process of vascular calcification, focusing specifically on the discrete and synergistic effects of calcium and phosphate in mediating vascular smooth muscle cell apoptosis, osteochondrocytic differentiation, vesicle release, calcification inhibitor expression, senescence, and death. Using our model of intact human vessels, factors initiating vascular calcification in vivo and the role of calcium and phosphate in driving accelerated calcification ex vivo are described. This work allows us to link clinical and basic research into a working theoretical model to explain the pathway of development of vascular calcification in CKD.

PLoS one 7(8), e43229 (2012). doi:10.1371/journal.pone.0043229, Published by PLoS [u.a.], Lawrence, Kan.

The structures of the M110 and M21 regulatory subunits of protein phosphatase-1M, the major enzyme which dephosphorylates myosin in smooth muscle, have been deduced from cloned cDNAs. The N-terminus of the M110 subunit from rat aorta contains seven ankyrin repeats, while the C-terminus of the M21 subunit from chicken gizzard contains a leucine zipper motif. The M110 subunit is expressed in two different forms which differ in their C-terminal sequences. One of these is highly homologous to the whole of the M21 subunit.

Human vascular smooth muscle cells (VSMCs) in culture are an important tool in understanding how VSMCs function and contribute to vessel wall contraction as well as disease. In this chapter, we describe methodologies that enable the investigator to culture large numbers of proliferative VSMCs. These VSMCs are heterogeneous and vary in size, shape, and proliferative capacity depending on the disease state and location of the vessel of origin. Therefore, we also describe techniques to validate their identity as bone fide VSMCs. Briefly, the methods include information on how to dissect the blood vessel to remove the medial layer containing VSMCs, as well as methods on how to propagate these cells, by either allowing VSMCs to migrate from the explanted medial tissue or by enzymatically dispersing the cells from the tissue. Both methods are suitable for culturing VSMCs derived from most vessel types with modifications of the enzyme dispersal method suitable for the isolation of microvessel VSMCs. An important feature of VSMCs in culture is that they lose many of their in vivo contractile properties and so model disease-associated VSMCs in the vessel wall rather than a non-proliferative contractile cell. To overcome this limitation, we also describe alternate methods that enable the study of cultured VSMCs in their contractile state by allowing the VSMCs to remain within an intact vessel ring. Overall, these procedures enable the investigator to undertake a diverse array of experimental assays on cultured VSMCs.

Abnormal proliferation of VSMC is a feature of atheromatous plaques and is responsible for obstructive neointimal lesions at the sites of mechanical or immunological intimal damage. We have discussed approaches to the development of specific inhibitors of VSMC proliferation based firstly on studies of the mechanism of action of known inhibitors and secondly on the identification of genes which are unique to and necessary for VSMC proliferation. The effects of angiotensin converting enzyme inhibitors, heparin and hexamethylene bisacetamide on VSMC proliferation have been discussed. Each of these types of agent has been shown to inhibit VSMC proliferation in vitro and/or in vivo. Examination of the mechanisms by which these agents inhibit VSMC proliferation reveals that transforming growth factor beta is an important mediator of their action and that the processes of de-differentiation and proliferation are independently regulated. Studies aimed at identifying genes involved in VSMC proliferation are at an early stage but have already provided strong evidence to support the hypothesis that VSMC in the vessel wall are heterogeneous and contain a subpopulation of cells with an enhanced proliferative capacity. Identification of the genes expressed by these cells may allow specific inhibitors of VSMC proliferation to be developed and may shed light on the pathogenesis of neointimal lesions.

To isolate specific markers of both differentiated and proliferating vascular smooth muscle cells (VSMCs), we used the technique of differential cDNA screening using RNA from cultured rat aortic VSMCs. The tissue specificity of expression of all of the cDNAs isolated was determined by Northern analysis. We isolated seven distinct cDNAs that were more strongly expressed in freshly dispersed, differentiated, aortic VSMCs compared with dedifferentiated late-passage cells. These were the cDNAs for tropoelastin, a matrix protein; alpha-smooth muscle (SM) actin, gamma-SM actin, calponin, and phospholamban, which are all proteins associated with the contractile function of differentiated VSMCs; SM22 alpha, a smooth muscle-specific protein of unknown function, and CHIP28, a putative membrane channel protein that is not highly expressed in other SM tissues and may therefore be a new VSMC marker. Two cDNAs that were expressed preferentially in late-passage dedifferentiated VSMCs were also isolated. These were the cDNAs for osteopontin and matrix Gla protein (MGP). Like CHIP28, MGP was strongly expressed in aortic VSMCs but not in other types of tissues containing SM cells, suggesting that both have specific functions in vascular tissue. Osteopontin and MGP have both previously been isolated from developing bone. Their expression in proliferating VSMCs suggests that they may be involved in regulating the calcification that commonly occurs in vascular lesions. The set of cDNAs obtained extends the range of DNA probes that are available for identifying VSMCs and characterizing their phenotype in vivo by in situ hybridization. Therefore, they should aid in the analysis of gene expression during the development of vessel lesions.

Introduction Vascular calcification is considered to be a significant predictor of coronary heart disease. It is also associated with serious clinical complications such as left ventricular hypertrophy and calcific uremic arteriolopathy. Matrix vesicles and apoptotic bodies (AB’s) released by calcifying vascular smooth muscle cells (VSMC’s) form the nidus for calcification in the vessel wall. Electron beam computed tomography (EBCT), the gold standard for non-invasive imaging and quantitation of vascular calcification fails to provide any information at the molecular and cellular level. It cannot differentiate between intimal and medial calcification and the early phase of calcification is not revealed on computed tomography (CT) images. We have synthesised a series of novel bone-seeking complexes of 99m Tc and used them for the detection of the process of vascular calcification in rat models. Materials and method Two established bone seeking radiopharmaceutical 18 F-Fluoride and 99m Tc-Methylenediphosphonate (MDP), as well as two novel bisphosphonates, 99m Tc(CO) 3 -Dipicolylamine (DPA) Alendronate and 99m Tc-Nitrido Bis(dithiocarbamatebisphosphonate) [ 99m Tc-N(DTC-BP) 2 ] were used in the study. In-vitro binding measurement studies were performed to compare the binding efficiencies of the radiopharmaceuticals with minerals from intimal and medial calcifications, powdered equine bone, synthetic hydroxyapatite and AB’s isolated from calcifying VSMC’s. In-vivo imaging was performed on Sprague-Dawley (S-D) rats fed with a warfarin diet to induce calcification in the tunica-media. SPECT-CT and PET-CT scanning were done on week 1, 2 and 4 of the diet to monitor progression of disease. Results The in vitro binding efficiencies of radiopharmaceuticals to the minerals were ranked as: Medial Mineral: 99m Tc-DPA Ale > 99m Tc-N(DTC-BP) 2 > 18 F-Fluoride > 99m Tc-MDP. Intimal mineral: 99m Tc-DPA Ale > 18 F-Fluoride > 99m Tc-N(DTC-BP) 2 > 99m Tc-MDP. In-vivo imaging with SPECT-CT and PET-CT revealed uptake of radiotracers in the aorta of warfarin fat rats as early as week 1 of the diet, where as calcification on the CT images was observed on week 4 in the same animal. Conclusions The findings indicate that novel bone seeking radiopharmaceuticals can bind with minerals present in intimal and medial aortic calcification and AB’s released by calcifying VSMC’s. In-vivo imaging results show that SPECT and PET scanning with bone seeking radiotracers can detect the process of calcification earlier than CT.

Arterial stiffening caused by deposition of calcium phosphate salts in the vessel wall is common in patients with atherosclerosis, renal failure and diabetes and contributes to high cardiovascular morbidity and mortality in these groups. Medial calcification is mediated by vascular smooth muscle cells (VSMCs) which undergo phenotypic transitions and secrete matrix vesicles (MVs) that form the first nidus for mineralization. In this study we studied the mechanisms regulating MV secretion and compared them with circulating calcifying calciprotein complexes and exosomes. Phenotypic modulation of human aortic VSMCs was induced by TGF-β1 or PDGF-BB. MVs and extracellular vesicles were isolated from cell media or platelet-free plasma, by differential ultracentrifugation and compared using Nanosight LM-10, flow cytometry, immunogold labelling, transmission electron microscopy and western blotting. We found that MVs are secreted from multivesicular bodies and enriched with the exosomal markers, CD63, CD81, CD9 and MHC I. Exosome secretion was regulated by the exosome biogenesis regulator, sphingomyelin phosphodiesterase 3 (SMPD3), and inhibition of SMPD3 prevented VSMC calcification. Treatment of VSMCs in calcifying conditions and loss of the contractile phenotype were both associated with elevated exosome secretion and increased calcification. In agreement with our in vitro data, MVB-like structures were observed in VSMCs ex vivo as shown by EM analysis of human vessel rings. Immunohistochemical staining revealed that the exosome markers, CD63 and annexin A6 were present in the extracellular (ECM) at sites of vascular calcification in vivo . Importantly, only exosomes secreted by VSMCs in calcifying conditions contained amorphous calcium phosphate crystals as detected by EDX analysis. Nanosite analysis revealed that calcifying VSMC-derived exosomes are similar in size to non-calcifying exosomes (mode 136 +/- 3.6 nm and 147+/- 5.9 nm, correspondingly) and were similar to exosomes, isolated from the blood (Mode 146+/-14 nm). Calcifying exosomes were also distinct from calcium phosphate crystals (mode 107 +/- 7 nm) and fetuin-A containing calciprotein particles (mode 182 +/- 9.3 nm). Our data indicate that vascular calcification is mediated by VSMC-derived exosomes and that loss of the contractile VSMC phenotype and mineral imbalance promote calcification by enhanced exosome secretion. Calcifying exosomes are distinct from calcium phosphate crystals or calciprotein particles, and may be presented in the circulation where their levels may be a novel indicator of vascular calcification.

Mendelian susceptibility to mycobacterial disease (MSMD) is a rare primary immunodeficiency associated with clinical disease caused by weakly virulent mycobacterial species. Interferon gamma receptor 1 (IFN-gammaR1) deficiency is a genetic etiology of MSMD. We describe the clinical and genetic features of a 7-year-old Italian boy suffering from MSMD associated with a complex phenotype, including neonatal hyperglycemia, neuromuscular disease, and dysmorphic features. The child also developed necrotizing pneumonia caused by Rhodococcus equi. The child is homozygous for a nonsense mutation in exon 3 of IFNGR1 as a result of paternal uniparental disomy (UPD) of the entire chromosome 6. This is the first reported case of uniparental disomy resulting in a complex phenotype including MSMD.

The Publisher's final version can be found by following the DOI link. The nuclear envelope (NE) LINC complex, in mammals comprised of SUN domain and nesprin proteins, provides a direct connection between the nuclear lamina and the cytoskeleton, which contributes to nuclear positioning and cellular rigidity. SUN1 and SUN2 interact with lamin A, but lamin A is only required for NE localization of SUN2, and it remains unclear how SUN1 is anchored. Here, we identify emerin and short nesprin-2 isoforms as novel nucleoplasmic binding partners of SUN1/2. These have overlapping binding sites distinct from the lamin A binding site. However, we demonstrate that tight association of SUN1 with the nuclear lamina depends upon a short motif within residues 209-228, a region that does not interact significantly with known SUN1 binding partners. Moreover, SUN1 localizes correctly in cells lacking emerin. Importantly then, the major determinant of SUN1 NE localization has yet to be identified. We further find that a subset of lamin A mutations, associated with laminopathies Emery-Dreifuss muscular dystrophy (EDMD) and Hutchinson-Gilford progeria syndrome (HGPS), disrupt lamin A interaction with SUN1 and SUN2. Despite this, NE localization of SUN1 and SUN2 is not impaired in cell lines from either class of patients. Intriguingly, SUN1 expression at the NE is instead enhanced in a significant proportion of HGPS but not EDMD cells and strongly correlates with pre-lamin A accumulation due to preferential interaction of SUN1 with pre-lamin A. We propose that these different perturbations in lamin A-SUN protein interactions may underlie the opposing effects of EDMD and HGPS mutations on nuclear and cellular mechanics.

Nuclear and cytoplasmic scaffold proteins have been shown to be essential for temporal and spatial organization, as well as the fidelity, of MAPK signaling pathways. In this study we show that nesprin-2 is a novel extracellular signal-regulated MAPK1 and 2 (ERK1/2) scaffold protein that serves to regulate nuclear signaling by tethering these kinases at promyelocytic leukemia protein nuclear bodies (PML NBs). Using immunofluorescence microscopy, GST pull-down and immunoprecipitation, we show that nesprin-2, ERK1/2, and PML colocalize and bind to form a nuclear complex. Interference of nesprin-2 function, by either siRNA-mediated knockdown or overexpression of a dominant negative nesprin-2 fragment, augmented ERK1/2 nuclear signaling shown by increased SP1 activity and ELK1 phosphorylation. The functional outcome of nesprin-2 disruption and the resultant sustained ERK1/2 signal was increased proliferation. Importantly, these activities were not induced by previously identified nuclear envelope (NE)-targeted nesprin-2 isoforms but rather were mediated by novel nuclear isoforms that lacked the KASH domain. Taken together, this study suggests that nesprin-2 is a novel intranuclear scaffold, essential for nuclear ERK1/2 signaling fidelity and cell cycle progression.

Widespread vascular calcification is a ubiquitous feature of aging and is prevalent in association with a number of common pathologies including atherosclerosis, renal failure, and diabetes. Once thought of as innocuous, emerging evidence suggests that calcification is causal in precipitating vascular events and mediating chronic cardiovascular damage, independent of disease context. Importantly, a large body of data has shed light on the factors that favor the formation of calcification in vivo, as well as on the complex mechanisms that initiate and promote it. This has identified some novel targets and allowed for the possibility that calcification can potentially be blocked and ultimately regressed. Targets include local and circulating inhibitors of calcification as well as factors that may ameliorate vascular smooth muscle cell (VSMC) apoptosis. Despite this, the vasculature remains a difficult tissue to target and currently there are no effective treatments in general use. More crucially, any potential treatments will need to be carefully evaluated as they may impinge on bone metabolism. Our best hope for the near future is to normalize factors associated with accelerated calcification in pathologies such as renal failure where, aberrant mineral metabolism, as well as treatment regimes, may contribute to the initiation and progression of calcification.

Background— Vascular calcification is associated with increased morbidity and mortality in stage V chronic kidney disease, yet its early pathogenesis and initiating mechanisms in vivo remain poorly understood. To address this, we quantified the calcium (Ca) load in arteries from children (10 predialysis, 24 dialysis) and correlated it with clinical, biochemical, and vascular measures. Methods and Results— Vessel Ca load was significantly elevated in both predialysis and dialysis and was correlated with the patients’ mean serum Ca×phosphate product. However, only dialysis patients showed increased carotid intima-media thickness and increased aortic stiffness, and calcification on computed tomography was present in only the 2 patients with the highest Ca loads. Importantly, predialysis vessels appeared histologically intact, whereas dialysis vessels exhibited evidence of extensive vascular smooth muscle cell (VSMC) loss owing to apoptosis. Dialysis vessels also showed increased alkaline phosphatase activity and Runx2 and osterix expression, indicative of VSMC osteogenic transformation. Deposition of the vesicle membrane marker annexin VI and vesicle component mineralization inhibitors fetuin-A and matrix Gla-protein increased in dialysis vessels and preceded von Kossa positive overt calcification. Electron microscopy showed hydroxyapatite nanocrystals within vesicles released from damaged/dead VSMCs, indicative of their role in initiating calcification. Conclusions— Taken together, this study shows that Ca accumulation begins predialysis, but it is the induction of VSMC apoptosis in dialysis that is the key event in disabling VSMC defense mechanisms and leading to overt calcification, eventually with clinically detectable vascular damage. Thus the identification of factors that lead to VSMC death in dialysis will be of prime importance in preventing vascular calcification.