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Physiological calcification plays an essential part in the development of the skeleton and teeth; however, the occurrence of calcification in soft tissues such as the brain, heart, and kidneys associates with health impacts, creating a massive social and economic burden. The current paradigm for pathological calcification focuses on the biological factors responsible for bone‐like mineralization, including osteoblast‐like cells and proteins inducing nucleation and crystal growth. However, the exact mechanism responsible for calcification remains unknown. Toward this goal, this review dissects the current understanding of structure–function relationships and physico‐chemical properties of pathologic calcification from a materials science point of view. We will discuss a range of potential mechanisms of pathological calcification, with the purpose of identifying universal mechanistic pathways that occur across multiple organs/tissues at multiple length scales. The possible effect of extracellular components in signaling and templating mineralization, as well as the role of intrinsically disordered proteins in calcification, is reviewed. The state‐of‐the‐art in vitro models and strategies that can recreate the highly dynamic environment of calcification are identified.

Supplemental Digital Content is available in the text., Objective: Vascular calcification is common among aging populations and mediated by vascular smooth muscle cells (VSMCs). The endoplasmic reticulum (ER) is involved in protein folding and ER stress has been implicated in bone mineralization. The role of ER stress in VSMC-mediated calcification is less clear. Approach and Results: mRNA expression of the ER stress markers PERK (PKR (protein kinase RNA)-like ER kinase), ATF (activating transcription factor) 4, ATF6, and Grp78 (glucose-regulated protein, 78 kDa) was detectable in human vessels with levels of PERK decreased in calcified plaques compared to healthy vessels. Protein deposition of Grp78/Grp94 was increased in the matrix of calcified arteries. Induction of ER stress accelerated human primary VSMC-mediated calcification, elevated expression of some osteogenic markers (Runx2 [RUNX family transcription factor 2], OSX [Osterix], ALP [alkaline phosphatse], BSP [bone sialoprotein], and OPG [osteoprotegerin]), and decreased expression of SMC markers. ER stress potentiated extracellular vesicle (EV) release via SMPD3 (sphingomyelin phosphodiesterase 3). EVs from ER stress-treated VSMCs showed increased Grp78 levels and calcification. Electron microscopy confirmed the presence of Grp78/Grp94 in EVs. siRNA (short interfering RNA) knock-down of Grp78 decreased calcification. Warfarin-induced Grp78 and ATF4 expression in rat aortas and VSMCs and increased calcification in an ER stress-dependent manner via increased EV release. Conclusions: ER stress induces vascular calcification by increasing release of Grp78-loaded EVs. Our results reveal a novel mechanism of action of warfarin, involving increased EV release via the PERK-ATF4 pathway, contributing to calcification. This study is the first to show that warfarin induces ER stress and to link ER stress to cargo loading of EVs.

Vascular calcification is a ubiquitous pathology of aging. Oxidative stress, persistent DNA damage, and senescence are major pathways driving both cellular and tissue aging, and emerging evidence suggests that these pathways are activated, and even accelerated, in patients with vascular calcification. The DNA damage response—a complex signaling platform that maintains genomic integrity—is induced by oxidative stress and is intimately involved in regulating cell death and osteogenic differentiation in both bone and the vasculature. Unexpectedly, a posttranslational modification, PAR (poly[ADP-ribose]), which is a byproduct of the DNA damage response, initiates biomineralization by acting to concentrate calcium into spheroidal structures that can nucleate apatitic mineral on the ECM (extracellular matrix). As we start to dissect the molecular mechanisms driving aging-associated vascular calcification, novel treatment strategies to promote healthy aging and delay pathological change are being unmasked. Drugs targeting the DNA damage response and senolytics may provide new avenues to tackle this detrimental and intractable pathology.

Dysregulated mineral metabolism and factors in uraemic serum place chronic kidney disease (CKD) patients at high risk of ectopic calcification, particularly vascular calcification. CKD patients have impaired phosphate excretion accompanied by klotho deficiency and vitamin D deficiency. The parathyroid hormone and fibroblast growth factor 23 negative feedback loops are ineffective and over activated leading to secondary hyperparathyroidism, CKD bone mineral disorder and a chronic increase in calcium (Ca) and phosphate (P). High serum Ca and P drive several mechanisms of calcification including apoptosis, osteogenic differentiation and the accumulation of both extracellular vesicles and calcium phosphate-containing particles (CPPs). Furthermore, the accumulation of uraemic toxins in serum has been shown to increase both oxidative and endoplasmic reticulum stress, which activate the inflammatory and DNA damage response pathways and promote calcification. There is no cure for vascular calcification, and treatment strategies involve preventing mineral dysregulation or directly targeting individual mechanisms of calcification.

Prevention of medial calcification in patients with chronic kidney disease requires the maintenance of vascular smooth muscle cell fitness. To preserve viability under chronic kidney disease–induced stress, vascular smooth muscle cells increase exosome formation and release, but the result is aggravated pathological calcification. Now Chen et al. report that microvesicles from calcifying vascular smooth muscle cells may propagate procalcifying signals to normal vascular smooth muscle cells. To help design effective strategies to impair procalcifying cell-to-cell communication, this commentary updates the current understanding of the main regulators of microvesicle/exosome biogenesis and secretion. Fil: Dusso, Adriana. Instituto de Investigación Sanitaria del Principado de Asturias; España Fil: Colombo, Maria Isabel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; Argentina Fil: Shanahan, Catherine M.. King’s College London; Reino Unido

Summary Biomineralization of the extracellular matrix is an essential, regulated process. Inappropriate mineralization of bone and the vasculature has devastating effects on patient health, yet an integrated understanding of the chemical and cell biological processes that lead to mineral nucleation remains elusive. Here, we report that biomineralization of bone and the vasculature is associated with extracellular poly(ADP-ribose) synthesized by poly(ADP-ribose) polymerases in response to oxidative and/or DNA damage. We use ultrastructural methods to show poly(ADP-ribose) can form both calcified spherical particles, reminiscent of those found in vascular calcification, and biomimetically calcified collagen fibrils similar to bone. Importantly, inhibition of poly(ADP-ribose) biosynthesis in vitro and in vivo inhibits biomineralization, suggesting a therapeutic route for the treatment of vascular calcifications. We conclude that poly(ADP-ribose) plays a central chemical role in both pathological and physiological extracellular matrix calcification., Graphical Abstract, Highlights • Poly(ADP-ribose) is found close to ECM calcification in developing bone and arteries • Poly(ADP-ribose) is produced in response to oxidative stress and delivered to the ECM • Poly(ADP-ribose) forms dense liquid droplets with calcium ions • Inhibiting PARP enzyme activity blocks calcification in vitro and in vivo, Müller et al. investigate the physicochemical process of extracellular matrix calcification in both physiological (bone) and pathological (vascular calcification) contexts. They find that oxidative stress-induced poly(ADP-ribose) nucleates calcium phosphate mineral crystals on extracellular matrix substrates and that calcification is inhibited by poly(ADP-ribose) polymerase (PARP) enzyme inhibitors.

Vascular smooth muscle cell (VSMC) phenotypic conversion from a contractile to 'synthetic' state contributes to vascular pathologies including restenosis, atherosclerosis and vascular calcification. We have recently found that the secretion of exosomes is a feature of 'synthetic' VSMCs and that exosomes are novel players in vascular repair processes as well as pathological vascular thrombosis and calcification. Pro-inflammatory cytokines and growth factors as well as mineral imbalance stimulate exosome secretion by VSMCs, most likely by the activation of sphingomyelin phosphodiesterase 3 (SMPD3) and cytoskeletal remodelling. Calcium stress induces dramatic changes in VSMC exosome composition and accumulation of phosphatidylserine (PS), annexin A6 and matrix metalloproteinase-2, which converts exosomes into a nidus for calcification. In addition, by presenting PS, VSMC exosomes can also provide the catalytic surface for the activation of coagulation factors. Recent data showing that VSMC exosomes are loaded with proteins and miRNA regulating cell adhesion and migration highlight VSMC exosomes as potentially important communication messengers in vascular repair. Thus, the identification of signalling pathways regulating VSMC exosome secretion, including activation of SMPD3 and cytoskeletal rearrangements, opens up novel avenues for a deeper understanding of vascular remodelling processes.

Background Ageing is a major risk factor for several vascular pathologies including calcification, the deposition of hydroxyapatite crystals in the vessel wall, and amyloid deposition. Exosomes, 20–100nm secreted vesicles, have been recognised as key mediators of amyloid formation in the brain and exosomes secreted from vascular smooth muscle cells (VSMCs) have been implicated in vascular calcification. However, whether exosomes link calcification and amyloid deposition in the vasculature is unknown. Medin, forms the age-related aortic medial amyloid (AMA) and is present in the aorta of 97% of people over 50-years old. Medin is a 50 amino acid cleavage product of the protein MFGE8 however, the mechanism of AMA formation is unknown. The aims of this study were to examine 1) changes in exosome secretion and content with age, 2) amyloid protein secretion and deposition by exosomes and 3) if calcifying conditions contribute to AMA formation. Methods Western blotting, qPCR, and immunostaining were used to study medin and MFGE8 expression in primary human VSMCs, at different ages and in elevated calcium and phosphate conditions. FACS was used for quantification of exosome secretion. Exosomes were isolated by differential ultracentrifugation. Extracellular matrix (ECM) was synthesised in vitro for immunofluorescence and Western blotting. Cresolphthalein assays were used to quantify calcification. Results MFGE8, medin and calcification, shown by von Kossa staining, co-localised in the medial layer of aortas from old subjects (figure 1). Medin and calcium deposits appear predominantly extracellular. Exosome secretion is increased from senescent VSMCs and in calcifying conditions. MFGE8 was expressed by VSMCs and was secreted exclusively in exosomes and its expression and secretion increased in calcifying conditions. Immunofluorescence staining of the ECM in vitro showed medin deposits co-localise with CD63, an exosome marker. The size of medin deposits in the ECM was increased when cells were treated with calcium and phosphate and this effect was reduced with an exosome secretion inhibitor, spiroepoxide (figure 2). Treatment with spiroepoxide also decreased hydroxyapatite deposition in the ECM. Conclusion Medin and MFGE8 are abundant in aged aortas and secreted by exosomes. The von Kossa and medin staining observed in the medial layer of aged subjects indicates co-localisation of AMA and calcification. Exosome secretion is increased with age as well as in calcifying conditions, which could contribute to the age-related deposition of medin in the ECM. Blocking exosome release attenuates the effect of elevated calcium and phosphate on both the size of medin aggregates and the formation of hydroxyapatite in the ECM. This suggests VSMC-derived exosomes mediate both vascular calcification and medin aggregation; and conditions which induce vascular calcification also stimulate AMA formation. Conflict of interest No conflict of interest

Magnesium has been shown to effectively prevent vascular calcification associated with chronic kidney disease. Magnesium has been hypothesized to prevent the upregulation of osteoblastic genes that potentially drives calcification. However, extracellular effects of magnesium on hydroxyapatite formation are largely neglected. This study investigated the effects of magnesium on intracellular changes associated with transdifferentiation and extracellular crystal formation. Bovine vascular smooth muscle cells were calcified using β-glycerophosphate. Transcriptional analysis, alkaline phosphatase activity and detection of apoptosis were used to identify transdifferentiation. Using X-ray diffraction and energy dispersive spectroscopy extracellular crystal composition was investigated. Magnesium prevented calcification in vascular smooth muscle cells. β-glycerophosphate increased expression of osteopontin but no other genes related to calcification. Alkaline phosphatase activity was stable and apoptosis was only detected after calcification independent of magnesium. Blocking of the magnesium channel TRPM7 using 2-APB did not abrogate the protective effects of magnesium. Magnesium prevented the formation of hydroxyapatite, which formed extensively during β-glycerophosphate treatment. Magnesium reduced calcium and phosphate fractions of 68% and 41% extracellular crystals, respectively, without affecting the fraction of magnesium. This study demonstrates that magnesium inhibits hydroxyapatite formation in the extracellular space, thereby preventing calcification of vascular smooth muscle cells.

Rationale: Matrix vesicles (MVs), secreted by vascular smooth muscle cells (VSMCs), form the first nidus for mineralization and fetuin-A, a potent circulating inhibitor of calcification, is specifically loaded into MVs. However, the processes of fetuin-A intracellular trafficking and MV biogenesis are poorly understood. Objective: The objective of this study is to investigate the regulation, and role, of MV biogenesis in VSMC calcification. Methods and Results: Alexa488-labeled fetuin-A was internalized by human VSMCs, trafficked via the endosomal system, and exocytosed from multivesicular bodies via exosome release. VSMC-derived exosomes were enriched with the tetraspanins CD9, CD63, and CD81, and their release was regulated by sphingomyelin phosphodiesterase 3. Comparative proteomics showed that VSMC-derived exosomes were compositionally similar to exosomes from other cell sources but also shared components with osteoblast-derived MVs including calcium-binding and extracellular matrix proteins. Elevated extracellular calcium was found to induce sphingomyelin phosphodiesterase 3 expression and the secretion of calcifying exosomes from VSMCs in vitro, and chemical inhibition of sphingomyelin phosphodiesterase 3 prevented VSMC calcification. In vivo, multivesicular bodies containing exosomes were observed in vessels from chronic kidney disease patients on dialysis, and CD63 was found to colocalize with calcification. Importantly, factors such as tumor necrosis factor-α and platelet derived growth factor-BB were also found to increase exosome production, leading to increased calcification of VSMCs in response to calcifying conditions. Conclusions: This study identifies MVs as exosomes and shows that factors that can increase exosome release can promote vascular calcification in response to environmental calcium stress. Modulation of the exosome release pathway may be as a novel therapeutic target for prevention.

Objective— The drug warfarin blocks carboxylation of vitamin K–dependent proteins and acts as an anticoagulant and an accelerant of vascular calcification. The calcification inhibitor MGP (matrix Gla [carboxyglutamic acid] protein), produced by vascular smooth muscle cells (VSMCs), is a key target of warfarin action in promoting calcification; however, it remains unclear whether proteins in the coagulation cascade also play a role in calcification. Approach and Results— Vascular calcification is initiated by exosomes, and proteomic analysis revealed that VSMC exosomes are loaded with Gla-containing coagulation factors: IX and X, PT (prothrombin), and proteins C and S. Tracing of Alexa488-labeled PT showed that exosome loading occurs by direct binding to externalized phosphatidylserine (PS) on the exosomal surface and by endocytosis and recycling via late endosomes/multivesicular bodies. Notably, the PT Gla domain and a synthetic Gla domain peptide inhibited exosome-mediated VSMC calcification by preventing nucleation site formation on the exosomal surface. PT was deposited in the calcified vasculature, and there was a negative correlation between vascular calcification and the levels of circulating PT. In addition, we found that VSMC exosomes induced thrombogenesis in a tissue factor–dependent and PS-dependent manner. Conclusions— Gamma-carboxylated coagulation proteins are potent inhibitors of vascular calcification suggesting warfarin action on these factors also contributes to accelerated calcification in patients receiving this drug. VSMC exosomes link calcification and coagulation acting as novel activators of the extrinsic coagulation pathway and inducers of calcification in the absence of Gla-containing inhibitors.

Contains fulltext : 177808.pdf (Publisher’s version ) (Closed access) Over the last decade, an increasing number of studies report a close relationship between serum magnesium concentration and cardiovascular disease risk in the general population. In end-stage renal disease, an association was found between serum magnesium and survival. Hypomagnesemia was identified as a strong predictor for cardiovascular disease in these patients. A substantial body of in vitro and in vivo studies has identified a protective role for magnesium in vascular calcification. However, the precise mechanisms and its contribution to cardiovascular protection remain unclear. There are currently 2 leading hypotheses: first, magnesium may bind phosphate and delay calcium phosphate crystal growth in the circulation, thereby passively interfering with calcium phosphate deposition in the vessel wall. Second, magnesium may regulate vascular smooth muscle cell transdifferentiation toward an osteogenic phenotype by active cellular modulation of factors associated with calcification. Here, the data supporting these major hypotheses are reviewed. The literature supports both a passive inorganic phosphate-buffering role reducing hydroxyapatite formation and an active cell-mediated role, directly targeting vascular smooth muscle transdifferentiation. However, current evidence relies on basic experimental designs that are often insufficient to delineate the underlying mechanisms. The field requires more advanced experimental design, including determination of intracellular magnesium concentrations and the identification of the molecular players that regulate magnesium concentrations in vascular smooth muscle cells. 01 augustus 2017

The nature of arterial changes resulting in cardiovascular events and dialysis vascular access failures in adult predialysis patients is not well known. This study examined intimal changes, calcium deposition, and consequent stiffness in brachial and radial arteries of adult CKD patients. Ten brachial-artery and seven radial-artery specimens were obtained during fistula creation from nine predialysis and eight dialysis-dependent, nondiabetic patients; and age-gender matched controls undergoing coronary bypass grafts (6 radial) or kidney donation (6 renal). Arterial stiffness was measured at baseline. Vessel histology, morphometric analysis of intima-media, and direct quantification of calcium load was performed using standard techniques. Both predialysis and dialysis patients demonstrated significant arterial intimal hyperplasia with intima:media ratio higher than controls (0.13 ± 0.12 vs. 0.02 ± 0.05, p = 0.01). Calcium deposition was demonstrated on histology and the calcium content in patients was higher than controls (34.68 ± 26.86 vs. 10.95 ± 9.18 μg/μg, p = 0.003). The blood vessel calcium content correlated with arterial stiffness (r = 0.64, p = 0.018). This study for the first time describes, and suggests mechanistic linkage between, intimal hyperplasia, pathological calcium deposition, and increased functional arterial stiffness in dialysis and predialysis patients. Our research could serve as a unique window into the in vivo status of the uremic vasculature impacting fistula maturation and cardiovascular disease.

Ageing is a potent, independent risk factor for cardiovascular disease. Calcification of the vascular smooth muscle cell (VSMC) layer of the vessel media is a hallmark of vascular ageing. Young patients with chronic kidney disease (CKD) exhibit an extremely high cardiovascular mortality, equivalent to that seen in octogenarians in the general population. Even children on dialysis develop accelerated medial vascular calcification and arterial stiffening, leading to the suggestion that patients with CKD exhibit a 'premature ageing' phenotype. It is now well documented that uraemic toxins, particularly those associated with dysregulated mineral metabolism, can drive VSMC damage and phenotypic changes that promote vascular calcification; epidemiological data suggest that some of these same risk factors associate with cardiovascular mortality in the aged general population. Importantly, emerging evidence suggests that uraemic toxins may promote DNA damage, a key factor driving cellular ageing, and moreover, that these ageing mechanisms may reiterate some of those seen in patients with genetically induced progeric syndromes caused by nuclear lamina disruption. This new knowledge should pave the way for the development of novel therapies that target tissue-specific ageing mechanisms to treat vascular decline in CKD.

Pathomechanisms underlying vascular calcification biogenesis are still incompletely understood. Biomineral from human atherosclerotic intimal plaques; human, equine, and bovine medial vascular calcifications; and human and equine bone was released from collagenous organic matrix by sodium hydroxide/sodium hypochlorite digestion. Solid-state (13)C NMR of intimal plaque mineral shows signals from cholesterol/cholesteryl esters and fatty acids. In contrast, in mineral from pure medial calcifications and bone mineral, fatty acid signals predominate. Refluxing (chloroform/methanol) intimal plaque calcifications removes the cholesterylic but not the fatty acyl signals. The lipid composition of this refluxed mineral now closely resembles that of the medial and bone mineral, which is unchanged by reflux. Thus, intimal and medial vascular calcifications and bone mineral have in common a pool of occluded mineral-entrained fatty acyl-rich lipids. This population of fatty acid may contain methyl-branched fatty acids, possibly representing lipoprotein particle remnants. Cell signaling and mechanistic parallels between physiological (orthotopic) and pathological (ectopic) calcification are also reflected thus in the NMR spectroscopic fingerprints of mineral-associated and mineral-entrained lipids. Additionally the atherosclerotic plaque mineral alone shows a significant independent pool of cholesterylic lipids. Colocalization of mineral and lipid may be coincidental, but it could also reflect an essential mechanistic component of biomineralization.

Vascular calcification is a pathological process common in patients with disorders of mineral metabolism and mediated by vascular smooth muscle cells (VSMCs). A key event in the initiation of VSMC calcification is the release of mineralization-competent matrix vesicles (MVs), small membrane-bound bodies with structural features enabling them to efficiently nucleate hydroxyapatite. These bodies are similar to MVs secreted by chondrocytes during bone development and their properties include the absence of calcification inhibitors, formation of nucleation sites, and accumulation of matrix metalloproteinases such as MMP-2. The mechanisms of MV biogenesis and loading remain poorly understood; however, emerging data have demonstrated that alterations in cytosolic calcium homeostasis can trigger multiple changes in MV composition that promote their mineralization.

Vascular calcification is an active and regulated process which is integral to cardiovascular disease and intimately linked to hypertension. Dysfunctional vascular smooth muscle cells, microvesicles, and dysregulated mineralization inhibitors play key roles in the calcification process, which occurs in the vessel intima in association with atherosclerosis as well as in the vessel media during ageing. Historically hypertension was considered a risk factor promoting atherosclerosis and associated intimal calcification. However, it is now recognized that not all vascular calcification occurs with atherosclerosis, and calcification of the vessel media is associated with arterial stiffening and is a major cause of isolated systolic hypertension in the elderly. Importantly, vascular calcification, regardless of its anatomical site, is an independent risk factor for cardiovascular mortality. Therefore, understanding the factors and mechanisms driving these processes will provide novel therapeutic targets for its prevention and perhaps ultimately its regression.

Rationale: Matrix vesicles (MVs) are specialized structures that initiate mineral nucleation during physiological skeletogenesis. Similar vesicular structures are deposited at sites of pathological vascular calcification, and studies in vitro have shown that elevated levels of extracellular calcium (Ca) can induce mineralization of vascular smooth muscle cell (VSMC)–derived MVs. Objectives: To determine the mechanisms that promote mineralization of VSMC-MVs in response to calcium stress. Methods and Results: Transmission electron microscopy showed that both nonmineralized and mineralized MVs were abundantly deposited in the extracellular matrix at sites of calcification. Using cultured human VSMCs, we showed that MV mineralization is calcium dependent and can be inhibited by BAPTA-AM. MVs released by VSMCs in response to extracellular calcium lacked the key mineralization inhibitor matrix Gla protein and showed enhanced matrix metalloproteinase-2 activity. Proteomics revealed that VSMC-MVs share similarities with chondrocyte-derived MVs, including enrichment of the calcium-binding proteins annexins (Anx) A2, A5, and A6. Biotin cross-linking and flow cytometry demonstrated that in response to calcium, AnxA6 shuttled to the plasma membrane and was selectively enriched in MVs. AnxA6 was also abundant at sites of vascular calcification in vivo, and small interfering RNA depletion of AnxA6 reduced VSMC mineralization. Flow cytometry showed that in addition to AnxA6, calcium induced phosphatidylserine exposure on the MV surface, thus providing hydroxyapatite nucleation sites. Conclusions: In contrast to the coordinated signaling response observed in chondrocyte MVs, mineralization of VSMC-MVs is a pathological response to disturbed intracellular calcium homeostasis that leads to inhibitor depletion and the formation of AnxA6/phosphatidylserine nucleation complexes.

Osteoprotegerin (OPG), a member of the TNF receptor superfamily, was initially found to modulate bone mass by blocking osteoclast maturation and function. Rodent models have also revealed a role for OPG as an inhibitor of vascular calcification. However, the precise mode of how OPG blocks mineralization is unclear. In this study, OPG was found in an in vitro assay to significantly inhibit calcification of vascular smooth muscle cells (VSMC) induced by high calcium/phosphate (Ca/P) treatment (p=0.0063), although this effect was blunted at high OPG concentrations. By confocal microscopy, OPG was detected in VSMC in the Golgi, the same localization seen in osteoblasts, which express OPG in bone. Treatment of VSMC by minerals (Ca, P, or both) induced OPG mRNA expression as assessed by real-time quantitative PCR, and VSMC derived from atherosclerotic plaque material also exhibited higher OPG expression as compared to control cells (p

Vascular calcification, which contributes to cardiovascular disease in patients with uremic hyperphosphatemia, is associated with vascular cell expression of osteogenic genes, including bone morphogenetic protein (BMP)-2 and osteopontin (OPN). High inorganic phosphate levels in vitro stimulate the osteogenic conversion of smooth muscle cells; however, the mechanism governing this is not clear. We found that high-phosphate medium increased the expression of BMP-2 and OPN in mouse smooth muscle cells in culture. However, this effect was lost in the presence of the mineralization inhibitor, pyrophosphate, suggesting a contribution of calcium phosphate crystals. Addition of 1–2 mmol/l phosphate alone to growth medium was sufficient to induce nanosized crystals after 1 day at 37 °C. Isolated crystals were about 160 nm in diameter and had a calcium to phosphate ratio of 1.35, consistent with the hydroxyapatite precursor octacalcium phosphate. Nanocrystal formation increased fourfold in the absence of serum, was blocked by fetuin-A, and was dependent on time and on the concentrations of phosphate and calcium. Purified synthetic hydroxyapatite nanocrystals and isolated high-phosphate-induced nanocrystals, but not nanocrystal-free high-phosphate medium, also induced BMP-2 and OPN. Thus, our results suggest that BMP-2 and OPN are induced by calcium phosphate nanocrystals, rather than soluble phosphate. This mechanism may contribute, in part, to hyperphosphatemia-related vascular cell differentiation and calcification.

Vascular stiffening and calcification are hallmarks of ageing and these pathologies are accelerated in patients with diabetes and renal failure. Emerging evidence has defined a role for nuclear lamina defects and the DNA damage response in driving vascular calcification. The mechanisms responsible are linked to the onset of cellular senescence and the activation of the proinflammatory senescence associated secretory phenotype (SASP) in vascular smooth muscle cells (VSMCs). In response to the SASP VSMCs can undergo phenotypic modulation and upregulate expression of proteins normally confined to cells of the osteo/chondrogenic lineage. Understanding the signaling pathways that drive this response is essential for defining novel therapeutic pathways to tackle age-associated vascular pathologies. We have been investigating the role of a number of signaling pathways involved in the DNA damage response as key drivers of calcification and VSMC osteo/chondrogenic differentiation including both the ataxia telangiectasia mutated (ATM) and poly(ADP) ribose (PARP) pathways. In addition it is clear that epigenetic changes, induced by nuclear lamina defects, precede the onset of calcification in both the aged vasculature and in patients with renal failure.

In chronic kidney disease (CKD) vascular calcification occurs in response to deranged calcium and phosphate metabolism and is characterized by vascular smooth muscle cell (VSMC) damage and attrition. To gain mechanistic insights into how calcium and phosphate mediate calcification, we used an ex vivo model of human vessel culture. Vessel rings from healthy control subjects did not accumulate calcium with long-term exposure to elevated calcium and/or phosphate. In contrast, vessel rings from patients with CKD accumulated calcium; calcium induced calcification more potently than phosphate (at equivalent calcium-phosphate product). Elevated phosphate increased alkaline phosphatase activity in CKD vessels, but inhibition of alkaline phosphatase with levamisole did not block calcification. Instead, calcification in CKD vessels most strongly associated with VSMC death resulting from calcium- and phosphate-induced apoptosis; treatment with a pan-caspase inhibitor ZVAD ameliorated calcification. Calcification in CKD vessels was also associated with increased deposition of VSMC-derived vesicles. Electron microscopy confirmed increased deposition of vesicles containing crystalline calcium and phosphate in the extracellular matrix of dialysis vessel rings. In contrast, vesicle deposition and calcification did not occur in normal vessel rings, but we observed extensive intracellular mitochondrial damage. Taken together, these data provide evidence that VSMCs undergo adaptive changes, including vesicle release, in response to dysregulated mineral metabolism. These adaptations may initially promote survival but ultimately culminate in VSMC apoptosis and overt calcification, especially with continued exposure to elevated calcium.

Objectives— Cell biological studies demonstrate remarkable similarities between mineralization processes in bone and vasculature, but knowledge of the components acting to initiate mineralization in atherosclerosis is limited. The molecular level microenvironment at the organic-inorganic interface holds a record of the mechanisms controlling mineral nucleation. This study was undertaken to compare the poorly understood interface in mineralized plaque with that of bone, which is considerably better characterized. Methods and Results— Solid state nuclear magnetic resonance (SSNMR) spectroscopy provides powerful tools for studying the organic-inorganic interface in calcium phosphate biominerals. The rotational echo double resonance (REDOR) technique, applied to calcified human plaque, shows that this interface predominantly comprises sugars, most likely glycosaminoglycans (GAGs). In this respect, and in the pattern of secondary effects seen to protein (mainly collagen), calcified plaque strongly resembles bone. Conclusion— The similarity between biomineral formed under highly controlled (bone) and pathological (plaque) conditions suggests that the control mechanisms are more similar than previously thought, and may be adaptive. It is strong further evidence for regulation of plaque mineralization by osteo/chondrocytic vascular smooth muscle cells.

Background. Vascular calcification occurs in the majority of patients with chronic kidney disease, but a subset of patients does not develop calcification despite exposure to a similar uraemic environment. Physiological inhibitors of calcification, fetuin-A, osteoprotegerin (OPG) and undercarboxylated-matrix Gla protein (uc-MGP) may play a role in preventing the development and progression of ectopic calcification, but there are scarce and conflicting data from clinical studies.Methods. We measured fetuin-A, OPG and uc-MGP in 61 children on dialysis and studied their associations with clinical, biochemical and vascular measures.Results. Fetuin-A and OPG were higher and uc-MGP lower in dialysis patients than controls. In controls, fetuin-A and OPG increased with age. Fetuin-A showed an inverse correlation with dialysis vintage (P = 0.0013), time-averaged serum phosphate (P = 0.03) and hs-CRP (P = 0.001). Aortic pulse wave velocity (PWV) and augmentation index showed a negative correlation with fetuin-A while a positive correlation was seen with PWV and OPG. Patients with calcification had lower fetuin-A and higher OPG than those without calcification. On multiple linear regression analysis Fetuin-A independently predicted aortic PWV (P = 0.004, ss = -0.45, model R-2 = 48%) and fetuin-A and OPG predicted cardiac calcification (P = 0.02, ss = -0.29 and P = 0.014, ss = 0.33, respectively, model R-2 = 32%).Conclusions. This is the first study to define normal levels of the calcification inhibitors in children and show that fetuin-A and OPG are associated with increased vascular stiffness and calcification in children on dialysis. Higher levels of fetuin-A in children suggest a possible protective upregulation of fetuin-A in the early stages of exposure to the pro-calcific and pro-inflammatory uraemic environment.

The layman’s term “hardening of the arteries” is synonymous with vascular disease and describes the ubiquitous calcification of the intima and media of the vessel wall that occurs in atherosclerosis and aging. Vascular calcification measured and quantified by electron beam computer tomography is a powerful predictor of myocardial infarction, vascular complications such as amputation, and all-cause mortality.1 Despite this fact, calcification has remained a neglected pathology, particularly in the field of atherosclerosis. This neglect is due in part to the controversy surrounding whether calcification is a bystander marker of disease load or acts to induce plaque rupture and thus increase the cardiovascular event rate; it is also due to the long-held belief that vascular calcification is an end-stage dystrophic process and therefore not amenable to manipulation. However, huge progress has been made over the last 10 years in elucidating the cell biological mechanisms underlying vascular calcification. Article p 2841 Consensus now exists that calcification is a regulated process, orchestrated by vascular smooth muscle cells (VSMCs) and occurring stepwise in a manner analogous to physiological mineralization processes. In the normal vessel wall, VSMCs are protected from calcification by local and circulating proteins such as matrix Gla protein and fetuin-A that act as mineralization inhibitors. However, in response to insults, VSMCs may die by apoptosis and release apoptotic bodies or may be induced by signals and mechanisms that are still not well defined to release matrix vesicles.2 These small membrane-bound microparticles have the capacity to concentrate calcium and phosphate to allow crystal nucleation and thus act as the first nidus for mineralization. Concomitant with vesicle release, although the precise order and relationship between these 2 events remain unclear, VSMCs undergo an osteo/chondrocytic phenotypic transition and begin to express transcription factors normally associated with differentiated chondrocytes and osteoblasts such as Sox …

Vascular calcification is an active, cell-mediated process that results from an imbalance between the promoters and inhibitors of mineralization. The process of vascular calcification shares many similarities with that of skeletal mineralization. However, while skeletal mineralization is a regulated process induced by complex, well-timed developmental cues, vascular calcification is a pathological process, occurring in response to dysregulated/inappropriate environmental cues. Damage inducing agents present in the uremic milieu such as a mineral imbalance, induce vascular smooth muscle cell (VSMC) apoptosis, and vesicle release resulting in mineral nucleation and the deposition of hydroxyapatite. Under normal conditions, inhibitors of soft-tissue mineralization such as matrix gamma-carboxyglutamic acid protein are expressed locally within the vessel wall while others such as fetuin-A are present in the circulation. Down-regulation or perturbation of these proteins leads to a phenotypic transformation of VSMC into osteo/chondrocytic-like cells that have the capacity to modulate the mineralization process. Many aspects of the mechanisms underlying vascular calcification have been defined through in vitro studies and molecular biological techniques; however, there are still unanswered questions, particularly with respect to the relationship between bone and vascular calcification, processes that appear to be inversely related. A better understanding of the complex mechanisms regulating tissue calcification may have therapeutic potential in reducing the cardiovascular disease-associated morbidity and mortality in patients with renal disease.

Patients with osteoporosis frequently suffer from vascular calcification, which was shown to predict both cardiovascular morbidity/mortality and osteoporotic fractures. Various common risk factors and mechanisms have been suggested to cause both bone loss and vascular calcification, including aging, estrogen deficiency, vitamin D and K abnormalities, chronic inflammation and oxidative stress. Major breakthroughs in molecular and cellular biology of bone metabolism and the characterization of knockout animals with deletion of bone-related genes have led to the concept that common signaling pathways, transcription factors and extracellular matrix interactions may account for both skeletal and vascular abnormalities. For example, mice that lack the cytokine decoy receptor osteoprotegerin or the hormone Klotho display a combined osteoporosis-arterial calcification phenotype. In this review, we summarize the current data and evaluate potential mechanisms of the osteoporosis-arterial calcification syndrome. We propose a unifying hypothesis of vascular calcification that combines both active and passive mechanisms of vascular mineralization with aspects of bone resorption and age-related changes.

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 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).

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.

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.

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 …

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 —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.

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.

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.

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.