Role of the extracellular ATP/pyrophosphate metabolism cycle in vascular calcification.

Conventionally, ATP is considered to be the principal energy source in cells. However, over the last few years, a novel role for ATP as a potent extracellular signaling molecule and the principal source of extracellular pyrophosphate, the main endogenous inhibitor of vascular calcification, has emerged. A large body of evidence suggests that two principal mechanisms are involved in the initiation and progression of ectopic calcification: high phosphate concentration and pyrophosphate deficiency. Pathologic calcification of cardiovascular structures, or vascular calcification, is a feature of several genetic diseases and a common complication of chronic kidney disease, diabetes, and aging. Previous studies have shown that the loss of function of several enzymes and transporters involved in extracellular ATP/pyrophosphate metabolism is associated with vascular calcification. Therefore, pyrophosphate homeostasis should be further studied to facilitate the design of novel therapeutic approaches for ectopic calcification of cardiovascular structures, including strategies to increase pyrophosphate concentrations by targeting the ATP/pyrophosphate metabolism cycle.

ATP-based therapy prevents vascular calcification and extends longevity in a mouse model of Hutchinson-Gilford progeria syndrome.

Pyrophosphate deficiency may explain the excessive vascular calcification found in children with Hutchinson-Gilford progeria syndrome (HGPS) and in a mouse model of this disease. The present study found that hydrolysis products of ATP resulted in a <9% yield of pyrophosphate in wild-type blood and aortas, showing that eNTPD activity (ATP → phosphate) was greater than eNPP activity (ATP → pyrophosphate). Moreover, pyrophosphate synthesis from ATP was reduced and pyrophosphate hydrolysis (via TNAP; pyrophosphate → phosphate) was increased in both aortas and blood obtained from mice with HGPS. The reduced production of pyrophosphate, together with the reduction in plasma ATP, resulted in marked reduction of plasma pyrophosphate. The combination of TNAP inhibitor levamisole and eNTPD inhibitor ARL67156 increased the synthesis and reduced the degradation of pyrophosphate in aortas and blood ex vivo, suggesting that these combined inhibitors could represent a therapeutic approach for this devastating progeroid syndrome. Treatment with ATP prevented vascular calcification in HGPS mice but did not extend longevity. By contrast, combined treatment with ATP, levamisole, and ARL67156 prevented vascular calcification and extended longevity by 12% in HGPS mice. These findings suggest a therapeutic approach for children with HGPS.

Vascular Calcification: Key Roles of Phosphate and Pyrophosphate.

Cardiovascular complications due to accelerated arterial stiffening and atherosclerosis are the leading cause of morbimortality in Western society. Both pathologies are frequently associated with vascular calcification. Pathologic calcification of cardiovascular structures, or vascular calcification, is associated with several diseases (for example, genetic diseases, diabetes, and chronic kidney disease) and is a common consequence of aging. Calcium phosphate deposition, mainly in the form of hydroxyapatite, is the hallmark of vascular calcification and can occur in the medial layer of arteries (medial calcification), in the atheroma plaque (intimal calcification), and cardiac valves (heart valve calcification). Although various mechanisms have been proposed for the pathogenesis of vascular calcification, our understanding of the pathogenesis of calcification is far from complete. However, in recent years, some risk factors have been identified, including high serum phosphorus concentration (hyperphosphatemia) and defective synthesis of pyrophosphate (pyrophosphate deficiency). The balance between phosphate and pyrophosphate, strictly controlled by several genes, plays a key role in vascular calcification. This review summarizes the current knowledge concerning phosphate and pyrophosphate homeostasis, focusing on the role of extracellular pyrophosphate metabolism in aortic smooth muscle cells and macrophages.

Vascular Smooth Muscle-Specific Progerin Expression Accelerates Atherosclerosis and Death in a Mouse Model of Hutchinson-Gilford Progeria Syndrome.

BACKGROUND: Progerin, an aberrant protein that accumulates with age, causes the rare genetic disease Hutchinson-Gilford progeria syndrome (HGPS). Patients who have HGPS exhibit ubiquitous progerin expression, accelerated aging and atherosclerosis, and die in their early teens, mainly of myocardial infarction or stroke. The mechanisms underlying progerin-induced atherosclerosis remain unexplored, in part, because of the lack of appropriate animal models. METHODS: We generated an atherosclerosis-prone model of HGPS by crossing apolipoprotein E-deficient (Apoe-/-) mice with LmnaG609G/G609G mice ubiquitously expressing progerin. To induce progerin expression specifically in macrophages or vascular smooth muscle cells (VSMCs), we crossed Apoe-/-LmnaLCS/LCS mice with LysMCre and SM22αCre mice, respectively. Progerin expression was evaluated by polymerase chain reaction and immunofluorescence. Cardiovascular alterations were determined by immunofluorescence and histology in male mice fed normal chow or a high-fat diet. In vivo low-density lipoprotein retention was assessed by intravenous injection of fluorescently labeled human low-density lipoprotein. Cardiac electric defects were evaluated by electrocardiography. RESULTS: Apoe-/-LmnaG609G/G609G mice with ubiquitous progerin expression exhibited a premature aging phenotype that included failure to thrive and shortened survival. In addition, high-fat diet-fed Apoe-/-LmnaG609G/G609G mice developed a severe vascular pathology, including medial VSMC loss and lipid retention, adventitial fibrosis, and accelerated atherosclerosis, thus resembling most aspects of cardiovascular disease observed in patients with HGPS. The same vascular alterations were also observed in Apoe-/-LmnaLCS/LCSSM22αCre mice expressing progerin specifically in VSMCs, but not in Apoe-/-LmnaLCS/LCSLysMCre mice with macrophage-specific progerin expression. Moreover, Apoe-/-LmnaLCS/LCSSM22αCre mice had a shortened lifespan despite the lack of any overt aging phenotype. Aortas of ubiquitously and VSMC-specific progerin-expressing mice exhibited increased retention of fluorescently labeled human low-density lipoprotein, and atheromata in both models showed vulnerable plaque features. Immunohistopathological examination indicated that Apoe-/-LmnaLCS/LCSSM22αCre mice, unlike Apoe-/-LmnaG609G/G609G mice, die of atherosclerosis-related causes. CONCLUSIONS: We have generated the first mouse model of progerin-induced atherosclerosis acceleration, and demonstrate that restricting progerin expression to VSMCs is sufficient to accelerate atherosclerosis, trigger plaque vulnerability, and reduce lifespan. Our results identify progerin-induced VSMC death as a major factor triggering atherosclerosis and premature death in HGPS.

Synthesis of Extracellular Pyrophosphate Increases in Vascular Smooth Muscle Cells During Phosphate-Induced Calcification.

Objective- Hydroxyapatite deposition on the medial layer of the aortic walls is the hallmark of vascular calcification and the most common complication in aging individuals and in patients with diabetes mellitus and those undergoing hemodialysis. Extracellular pyrophosphate is a potent physicochemical inhibitor of hydroxyapatite crystal formation. This study analyzed changes in extracellular pyrophosphate metabolism during the phosphate-induced calcification process. Approach and Results- Phosphate-induced calcification of ex vivo-cultured aortic rings resulted in calcium accumulation after 7 days. This accumulation was enhanced when aortic walls were devitalized. BMP2 (bone morphogenic protein 2) expression was associated with calcium accumulation in cultured aortic rings, as well as in cultured vascular smooth muscle cells (VSMCs) and in calcitriol-induced calcification in rats. Hydroxyapatite dose dependently induced BMP2 overexpression in VSMCs. Moreover, TNAP (tissue nonspecific alkaline phosphatase) mRNA levels and activity were found to be downregulated in early phases and upregulated in later phases of calcification in all 3 models studied. eNPP1 (ectonucleotide pyrophosphatase/phosphodiesterase 1) increased from early to later phases of calcification, whereas eNTPD1 (ectonucleoside triphosphate diphosphohydrolase 1) was downregulated during later phases. Synthesis of pyrophosphate in VSMCs increased significantly over time, in all 3 models studied. Because the rate of pyrophosphate hydrolysis was 10× slower than the rate of pyrophosphate synthesis, pyrophosphate synthesis is determined mainly by the ratio of eNPP1 to eNTPD1 activity. Hydroxyapatite also induces increments both in TNAP and eNPP1/eNTPD1 ratio in VSMCs. Conclusions- Pyrophosphate synthesis increases in VSMCs during phosphate-induced calcification because of compensatory regulation of extracellular pyrophosphate metabolism.

Pyrophosphate deficiency in vascular calcification.

Pathologic cardiovascular calcification is associated with a number of conditions and is a common complication of chronic kidney disease. Because ambient calcium and phosphate levels together with properties of the vascular matrix favor calcification even under normal conditions, endogenous inhibitors such as pyrophosphate play a key role in prevention. Genetic diseases and animal models have elucidated the metabolism of extracellular pyrophosphate and demonstrated the importance of pyrophosphate deficiency in vascular calcification. Therapies based on pyrophosphate metabolism have been effective in animal models, including renal failure, and hold promise as future therapies to prevent vascular calcification.

Alternatively activated macrophages exhibit an anticalcifying activity dependent on extracellular ATP/pyrophosphate metabolism.

Calcium-phosphate deposition (CPD) in atherosclerotic lesions, which begins in middle age and increases with aging, is a major independent predictor of future cardiovascular disease morbi-mortality. Remodeling of atherosclerotic vessels during aging is regulated in part by intimal macrophages, which can polarize to phenotypically distinct populations with distinct functions. This study tested the hypothesis that classically activated macrophages (M1φs) and alternatively activated macrophages (M2φs) differently affect vascular smooth muscle cell (VSMC) calcification and investigated the underlying mechanisms. We analyzed mouse VSMC-macrophage cocultures using a transwell system. Coculture of VSMCs with M2φs significantly reduced CPD, but coculture with M1φs had no effect. The anticalcific effect of M2φs was associated with elevated amounts of extracellular ATP and pyrophosphate (PPi), two potent inhibitors of CPD, and was lost upon forced hydrolysis of these metabolites. In M2φs and VSMC-M2φs cocultures, analysis of the ectoenzymes that regulate extracellular ATP/PPi metabolism revealed increased mRNA expression and activity of ectoenzyme nucleotide pyrophosphatase/phosphodiesterase-1, which synthesizes PPi from ATP, without changes in tissue-nonspecific alkaline phosphatase, which hydrolyzes PPi In conclusion, increased accumulation of extracellular ATP and PPi by alternatively activated mouse M2φs inhibits CPD. These results reveal novel mechanisms underlying macrophage-dependent control of intimal calcification.

Defective extracellular pyrophosphate metabolism promotes vascular calcification in a mouse model of Hutchinson-Gilford progeria syndrome that is ameliorated on pyrophosphate treatment.

BACKGROUND: Progerin is a mutant form of lamin A responsible for Hutchinson-Gilford progeria syndrome (HGPS), a premature aging disorder characterized by excessive atherosclerosis and vascular calcification that leads to premature death, predominantly of myocardial infarction or stroke. The goal of this study was to investigate mechanisms that cause excessive vascular calcification in HGPS. METHODS AND RESULTS: We performed expression and functional studies in wild-type mice and knock-in Lmna(G609G/+) mice expressing progerin, which mimic the main clinical manifestations of HGPS. Lmna(G609G/+) mice showed excessive aortic calcification, and primary aortic vascular smooth muscle cells from these progeroid animals had an impaired capacity to inhibit vascular calcification. This defect in progerin-expressing vascular smooth muscle cells is associated with increased expression and activity of tissue-nonspecific alkaline phosphatase and mitochondrial dysfunction, which leads to reduced ATP synthesis. Accordingly, Lmna(G609G/+) vascular smooth muscle cells are defective for the production and extracellular accumulation of pyrophosphate, a major inhibitor of vascular calcification. We also found increased alkaline phosphatase activity and reduced ATP and pyrophosphate levels in plasma of Lmna(G609G/+) mice without changes in phosphorus and calcium. Treatment with pyrophosphate inhibited vascular calcification in progeroid mice. CONCLUSIONS: Excessive vascular calcification in Lmna(G609G) mice is caused by reduced extracellular accumulation of pyrophosphate that results from increased tissue-nonspecific alkaline phosphatase activity and diminished ATP availability caused by mitochondrial dysfunction in vascular smooth muscle cells. Excessive calcification is ameliorated on pyrophosphate treatment. These findings reveal a previously undefined pathogenic process in HGPS that may also contribute to vascular calcification in normal aging, because progerin progressively accumulates in the vascular tissue of individuals without HGPS.

Vascular Calcification: A Passive Process That Requires Active Inhibition.

The primary cause of worldwide mortality and morbidity stems from complications in the cardiovascular system resulting from accelerated atherosclerosis and arterial stiffening. Frequently, both pathologies are associated with the pathological calcification of cardiovascular structures, present in areas such as cardiac valves or blood vessels (vascular calcification). The accumulation of hydroxyapatite, the predominant form of calcium phosphate crystals, is a distinctive feature of vascular calcification. This phenomenon is commonly observed as a result of aging and is also linked to various diseases such as diabetes, chronic kidney disease, and several genetic disorders. A substantial body of evidence indicates that vascular calcification involves two primary processes: a passive process and an active process. The physicochemical process of hydroxyapatite formation and deposition (a passive process) is influenced significantly by hyperphosphatemia. However, the active synthesis of calcification inhibitors, including proteins and low-molecular-weight inhibitors such as pyrophosphate, is crucial. Excessive calcification occurs when there is a loss of function in enzymes and transporters responsible for extracellular pyrophosphate metabolism. Current in vivo treatments to prevent calcification involve addressing hyperphosphatemia with phosphate binders and implementing strategies to enhance the availability of pyrophosphate.

Prevention of vascular calcification by polyphosphates and nucleotides- role of ATP.

BACKGROUND: In recent decades, the prevention of vascular calcification (VC) by pyrophosphate (PPi), bisphosphonates, and polyphosphates has been extensively reported. However, the possibility of direct inhibition of calcium phosphate deposition (CPD) by nucleoside-associated polyphosphates has not been addressed. We analyzed the role of ATP as an inhibitor of calcification in 2 ways: by characterizing the extracellular hydrolysis of ATP as source of PPi in the aorta, and by demonstrating the ability of ATP to prevent CPD by acting as a polyphosphate. METHODS AND RESULTS: In our study, both PPi and ATP hydrolysis in the rat aorta was kinetically characterized, thereby resulting in apparent Michaelis-Menten constants of 179 and 435 µmol/l, respectively, with the corresponding maximal velocities of 55.1 and 6,177 nmol·g(-1)·min(-1). According to these kinetic parameters, the theoretical PPi concentration in the aortic wall was 0.4-3.5 µmol/L (for an ATP concentration range of 0.1-1.0 µmol/L). In addition, we showed that nonhydrolyzable molecules are more efficient as CPD inhibitors than endogenous compounds, in accordance with the IC50 values: 1.2-2.4 µmol/L for bisphosphonates vs. 8.8 µmol/L for PPi, and 0.5-1.5 µmol/L for nonhydrolyzable ATP analogs vs. 3.2 µmol/L for ATP. CONCLUSIONS: Extracellular ATP can play an important role in the prevention of VC, not only as the source of PPi but also as a direct inhibitor of CPD.

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

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

Extracellular pyrophosphate metabolism and calcification in vascular smooth muscle.

Extracellular inorganic pyrophosphate (ePP(i)) is an important endogenous inhibitor of vascular calcification, but it is not known whether systemic or local vascular PP(i) metabolism controls calcification. To determine the role of ePP(i) in vascular smooth muscle, we identified the pathways responsible for ePP(i) production and hydrolysis in rat and mouse aortas and manipulated them to demonstrate their role in the calcification of isolated aortas in culture. Rat and mouse aortas contained mRNA for ectonucleotide pyrophosphatase/phosphodiesterases (NPP1-3), the putative PP(i) transporter ANK, and tissue-nonspecific alkaline phosphatase (TNAP). Synthesis of PP(i) from ATP in aortas was blocked by ß,γ-methylene-ATP, an inhibitor of NPPs. Aortas from mice lacking NPP1 (Enpp1(-/-)) did not synthesize PP(i) from ATP and exhibited increased calcification in culture. Although ANK-mediated transport of PP(i) could not be demonstrated in aortas, aortas from mutant (ank/ank) mice calcified more in culture than did aortas from normal (ANK/ANK) mice. Hydrolysis of PP(i) was reduced 25% by ß,γ-methylene-ATP and 50% by inhibition of TNAP. Hydrolysis of PP(i) was increased in cells overexpressing TNAP or NPP3 but not NPP1 and was not reduced in Enpp1(-/-) aortas. Overexpression of TNAP increased calcification of cultured aortas. The results show that smooth muscle NPP1 and TNAP control vascular calcification through effects on synthesis and hydrolysis of ePP(i), indicating an important inhibitory role of locally produced PP(i). Smooth muscle ANK also affects calcification, but this may not be mediated through transport of PP(i). NPP3 is identified as an additional pyrophosphatase that could influence vascular calcification.

Calcium phosphate deposition with normal phosphate concentration. -Role of pyrophosphate-.

BACKGROUND: Calcium phosphate deposition (CPD) is the hallmark of vascular smooth muscle cell (VSMC) calcification. CPD is a thermodynamically-favored process under physiological conditions. Hydroxyapatite, the most common calcium phosphate in calcified arteries, is passively formed during VSMC calcification, independently on any direct cellular activity. Furthermore, in recent years it has been demonstrated there is an anti-calcifying effect by extracellular pyrophosphate, an endogenous inhibitor of CPD, both in vitro and in vivo, which directly blocks hydroxyapatite formation. METHODS AND RESULTS: We have used the in vitro calcification model without cellular activity, by treating confluent rat aortic VSMC with paraformaldehyde. Fixed cells were incubated with the indicated media to obtain or inhibit calcification. The calcium content was determined colorimetrically. Calcification was observed after 3 weeks (21 days) using a physiological concentration of calcium (1.8 mmol/L) and phosphate (1 mmol/L). Calcium deposition was directly proportional to the amount of phosphate in the media, with a calcification rate of 3.5, 7.5, and 14.3 µg·cm⁻²·day⁻¹, using 1, 2, and 4 mmol/L of phosphate, respectively. Under physiological conditions, pyrophosphate inhibits CPD with an IC50 of ≍200 nmol/L. CONCLUSIONS: CPD occurs under a physiological concentration of calcium and phosphate, but this deposition is completely inhibited in the presence of a physiological concentration of pyrophosphate (3-5 µmol/L).

Compensatory regulation of the sodium/phosphate cotransporters NaPi-IIc (SCL34A3) and Pit-2 (SLC20A2) during Pi deprivation and acidosis.

The role of four Pi transporters in the renal handling of Pi was analyzed using functional and molecular methods. The abundance of NaPi-IIa, NaPi-IIc, and Pit-2 was increased by 100% in kidney from rats on a 0.1% Pi diet, compared to a 0.6% Pi diet. Pit-1 was not modified. Type II-mediated Pi uptake in Xenopus oocytes increased as the pH of the uptake medium increased, and the opposite occurred with Pit-1 and Pit-2. At pH 6.0, Pi uptake mediated through type II was approximately 10% of the uptake at pH 7.5, but the uptake through Pit-2 was 250% of the activity at pH 7.5. Real brush-border membrane vesicles (BBMV) responded to pH changes following the same pattern as type II transporters. Adaptation to a 0.1% Pi diet was accompanied by a 65% increase in the V (max) of BBMV Pi transport at pH 7.5, compared to a 0.6% Pi diet. The increase was only 11% at pH 6.0. Metabolic acidosis increased the expression of NaPi-IIc and Pit-2 in animals adapted to a low Pi diet, and phosphaturia was only observed in control diet animals. The combination of the pH effect, Pi adaptation, and metabolic acidosis suggests very modest involvement of Pit-2 in renal Pi handling. Real-time PCR and mathematical analyses of transport findings suggest that NaPi-IIa RNA accounts for 95% of all Pi transporters and that type II handles 97% of Pi transport at pH 7.5 and 60% of Pi transport at pH 6.0, depending on the pH and the physiological conditions.

Arsenate transport by sodium/phosphate cotransporter type IIb.

Arsenic is a metalloid that causes the dysfunction of critical enzymes, oxidative stress, and malignancies. In recent years several transporters of As(III) have been identified, including aquaglyceroporins (AQP) and multidrug resistance proteins (MRP). As(V) transport, however, has not been sufficiently studied because it has been assumed that arsenate is taken up by mammalian cells through inorganic phosphate (Pi) transporters. In this paper we have analyzed the role of Pi transporters in the uptake of arsenate by directly using (73)As(V) as a radiotracer in phosphate transporter-expressing Xenopus laevis oocytes. The affinities of Pi transporters for H(3)AsO(4) were lower than the affinities for Pi. NaPiIIa, NaPiIIc, Pit1, and Pit2 showed a K(m) for arsenate that was >1mM (i.e., at least ten times lower than the affinities for Pi). The NaPiIIb isoform showed the highest affinity for As(V) in mouse (57 microM), rat (51 microM), and human (9.7 microM), which are very similar to the affinities for Pi. Therefore, NaPiIIb can have a prominent role in the toxicokinetics of arsenic following oral exposure to freshwater or food contaminated with As(V).

Phosphonoformic acid prevents vascular smooth muscle cell calcification by inhibiting calcium-phosphate deposition.

OBJECTIVE: The role of inorganic phosphate in the pathogenesis of vascular calcification (VC) has been studied extensively in recent years. Phosphonoformic acid (PFA), an inhibitor of type II Pi transporters, has been traditionally used to study the involvement of Pi transport in VC, because PFA also prevents calcium deposition in vitro. However, aortic vascular smooth muscle cells (VSMCs) only express PFA-resistant, type III transporters (Pit-1 and Pit-2). Therefore, in this article we have studied the mechanism of VC prevention by PFA. METHODS AND RESULTS: Radiotracer Pi uptake in rat VSMCs was not inhibited at the concentrations at which PFA prevents calcification. Alternative mechanisms whereby PFA could prevent calcification, such as cytotoxicity or phosphodiesterase inhibition, have also been excluded. The progression of calcification also took place in fixed cells. The kinetics of VC prevention by PFA, pyrophosphate, phosphonoacetate, and bisphosphonates was similar in live and fixed cells, showing mean effective concentrations in the micromolar range. CONCLUSIONS: PFA mainly prevents VC through a physicochemical mechanism that is independent of any cellular metabolic activity, including Pi transport. Conversely, PFA seems to act similarly to its chemical analogues, inorganic pyrophosphate, and bisphosphonates, as suggested decades ago.

Dietary magnesium supplementation improves lifespan in a mouse model of progeria.

Aging is associated with redox imbalance according to the redox theory of aging. Consistently, a mouse model of premature aging (LmnaG609G/+ ) showed an increased level of mitochondrial reactive oxygen species (ROS) and a reduced basal antioxidant capacity, including loss of the NADPH-coupled glutathione redox system. LmnaG609G/+ mice also exhibited reduced mitochondrial ATP synthesis secondary to ROS-induced mitochondrial dysfunction. Treatment of LmnaG609G/+ vascular smooth muscle cells with magnesium-enriched medium improved the intracellular ATP level, enhanced the antioxidant capacity, and thereby reduced mitochondrial ROS production. Moreover, treatment of LmnaG609G/+ mice with dietary magnesium improved the proton pumps (complexes I, III, and IV), stimulated extramitochondrial NADH oxidation and enhanced the coupled mitochondrial membrane potential, and thereby increased H+ -coupled mitochondrial NADPH and ATP synthesis, which is necessary for cellular energy supply and survival. Consistently, magnesium treatment reduced calcification of vascular smooth muscle cells in vitro and in vivo, and improved the longevity of mice. This antioxidant property of magnesium may be beneficial in children with HGPS.

New insights into endogenous mechanisms of protection against arterial calcification.

Cardiovascular complications due to accelerated atherosclerosis and arterial stiffening are the leading cause of morbidity and mortality in the Western society. Both pathologies are frequently associated with vascular calcification. Deposits of calcium phosphate salts, mainly in form of hydroxyapatite, is the hallmark of vascular calcification. Calcification is frequently observed in atherosclerotic lesions (intimal calcification) associated with vascular smooth muscle cells (VSMCs) and macrophages. By contrast, medial calcification, occurring in the elastic region of the arteries, is almost exclusively associated with VSMCs, and is common in arteriosclerosis related to aging, diabetes, and chronic kidney disease. In extracellular fluids, a range of endogenous low- and high-molecular weight calcification inhibitors are present, including osteopontin, matrix-Gla proteins and Fetuin A. Moreover, pyrophosphate deficiency plays a key role in vascular calcification. Pyrophosphate is produced by extracellular hydrolysis of ATP and is degraded to phosphate by tissue non-specific alkaline phosphatase. Loss of function in the enzymes and transporters involved in the extracellular pyrophosphate metabolism leads to excessive deposition of calcium-phosphate salts. This review summarizes the current knowledge about endogenous mechanisms of protection against calcification in the aortic wall, focusing on the role of extracellular pyrophosphate metabolism in vascular smooth muscle cells and macrophages.