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Peripheral serotonin levels are associated with cardiovascular disease risk. We previously found that serum serotonin levels are higher in hyperlipidemic mice than wild-type mice. Evidence also suggests that serotonin regulates biomineralization, in that serotonin treatment augments TNF-a-induced matrix calcification of aortic valve interstitial cells and that a selective inhibitor of peripheral serotonin, LP533401, rescues bone loss induced by ovariectomy in mice. Thus, in the present study, we examined the effects of LP533401 on both skeletal bone mineral density (BMD) and aortic calcification in both young and older hyperlipidemic mice susceptible to calcific atherosclerosis and bone loss. By serial in vivo microCT imaging, we assessed BMD and aortic calcification of Apoe -/- mice fed an atherogenic (high cholesterol) diet alone or mixed with LP533401. Results show that in the young mice, LP533401 blunted skeletal bone loss in lumbar vertebrae but not in femurs. LP533401 also blunted the initial development of aortic calcification but not its progression. Echocardiographic analysis showed that LP533401 blunted both hyperlipidemia-induced cardiac hypertrophy and left ventricular dysfunction. In the older mice, LP533401 increased the BMD of lumbar vertebrae but not of femurs. The aortic calcification progressed in both controls and LP533401-treated mice, but, at post-treatment, LP533401-treated mice had significantly less aortic calcification than the controls. These findings suggest that LP533401 mitigates adverse effects of hyperlipidemia on skeletal and vascular tissues in site- and stage-dependent manners., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Background and Aims: Cardiovascular disease risk is associated with coronary artery calcification and is mitigated by regular exercise. Paradoxically, elite endurance athletes, who have low risk, are likely to have more coronary calcification, raising questions about the optimal level of activity., Methods: Female hyperlipidemic (Apoe -/- ) mice with baseline aortic calcification were subjected to high-speed (18.5 m/min), low-speed (12.5 m/min), or no treadmill exercise for 9 weeks. 18 F-NaF microPET/CT images were acquired at weeks 0 and 9, and echocardiography was performed at week 9., Results: In controls, aortic calcium content and density increased significantly. Exercise regimens did not alter the time-dependent increase in content, but the increase in mean density was blunted. Interestingly, the low-speed regimen significantly reduced 18 F-NaF uptake, a marker of surface area. Left ventricular (LV) systolic function was lower while LV diameter was greater in the low-speed group compared with controls or the high-speed group. In the low-speed group, vertebral bone density by CT decreased significantly, contrary to expectations. Male hyperlipidemic (Apoe -/- ) mice were fed a Western diet and also subjected to low-speed or no exercise followed by imaging at weeks 0 and 9. In males, exercise also did not alter the time-dependent increase in aortic calcification. Exercise did not affect 18 F-NaF uptake or bone mineral density, but it blunted the diet-induced LV hypertrophy seen in controls., Conclusions: These results suggest that, in mice, exercise has differential effects on aortic calcification, cardiac function, and skeletal bone mineral density., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper, (Copyright © 2023 Elsevier B.V. All rights reserved.)

Recent studies have found an association between high volumes of physical activity and increased levels of coronary artery calcification (CAC) among older male endurance athletes, yet the underlying mechanisms have remained largely elusive. Potential mechanisms include greater exposure to inflammatory cytokines, reactive oxygen species and oxidised low-density lipoproteins, as acute strenuous physical activity has been found to enhance their systemic release. Other possibilities include post-exercise elevations in circulating parathyroid hormone, which can modify the amount and morphology of calcific plaque, and long-term exposure to non-laminar blood flow within the coronary arteries during vigorous physical activity, particularly in individuals with pre-existing atherosclerosis. Further, although the association has only been identified in men, the role of testosterone in this process remains unclear. This brief review discusses the association between high-volume endurance exercise and CAC in older men, elaborates on the potential mechanisms underlying the increased calcification, and provides clinical implications and recommendations for those at risk., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2023. No commercial re-use. See rights and permissions. Published by BMJ.)

Purpose of Review: Lipids and lipoproteins have long been known to contribute to atherosclerosis and cardiovascular calcification. One theme of recent work is the study of lipoprotein (a) [Lp(a)], a lipoprotein particle similar to LDL-cholesterol that carries a long apoprotein tail and most of the circulating oxidized phospholipids., Recent Findings: In-vitro studies show that Lp(a) stimulates osteoblastic differentiation and mineralization of vascular smooth muscle cells, while the association of Lp(a) with coronary artery calcification continues to have varying results, possibly because of the widely varying threshold levels of Lp(a) chosen for association analyses. Another emerging area in the field of cardiovascular calcification is pathological endothelial-to-mesenchymal transition (EndMT), the process whereby endothelial cell transition into multipotent mesenchymal cells, some of which differentiate into osteochondrogenic cells and mineralize. The effects of lipids and lipoproteins on EndMT suggest that they modulate cardiovascular calcification through multiple mechanisms. There are also emerging trends in imaging of calcific vasculopathy, including: intravascular optical coherence tomography for quantifying plaque characteristics, PET with a radiolabeled NaF tracer, with either CT or MRI to detect coronary plaque vulnerability., Summary: Recent work in this field includes studies of Lp(a), EndMT, and new imaging techniques., (Copyright © 2022 Wolters Kluwer Health, Inc. All rights reserved.)

Vascular calcification, once considered a degenerative, end-stage, and inevitable condition, is now recognized as a complex process regulated in a manner similar to skeletal bone at the molecular and cellular levels. Since the initial discovery of bone morphogenetic protein in calcified human atherosclerotic lesions, decades of research have now led to the recognition that the regulatory mechanisms and the biomolecules that control cardiovascular calcification overlap with those controlling skeletal mineralization. In this review, we focus on key biomolecules driving the ectopic calcification in the circulation and their regulation by metabolic, hormonal, and inflammatory stimuli. Although calcium deposits in the vessel wall introduce rupture stress at their edges facing applied tensile stress, they simultaneously reduce rupture stress at the orthogonal edges, leaving the net risk of plaque rupture and consequent cardiac events depending on local material strength. A clinically important consequence of the shared mechanisms between the vascular and bone tissues is that therapeutic agents designed to inhibit vascular calcification may adversely affect skeletal mineralization and vice versa. Thus, it is essential to consider both systems when developing therapeutic strategies.

Purpose of Review: This review addresses recent developments in studies of lipid regulation of calcific disease of arteries and cardiac valves, including the role of nuclear receptors. The role of lipid-soluble signals and their receptors is timely given the recent evidence and concerns that lipid-lowering treatment may increase the rate of progression of coronary artery calcification, which has been long associated with increased cardiovascular risk. Understanding the mechanisms will be important for interpreting such clinical information., Recent Findings: New findings support regulation of calcific vascular and valvular disease by nuclear receptors, including the vitamin D receptor, glucocorticoid receptor, nutrient-sensing nuclear receptors (liver X receptor, farnesoid X receptor, and peroxisome proliferator-activated receptors), and sex hormone (estrogen and androgen) receptors. There were two major unexpected findings: first, vitamin D supplementation, which was previously believed to prevent or reduce vascular calcification, showed no cardiovascular benefit in large randomized, controlled trials. Second, both epidemiological studies and coronary intravascular ultrasound studies suggest that treatment with HMG-CoA reductase inhibitors increases progression of coronary artery calcification, raising a question of whether there are mechanically stable and unstable forms of coronary calcification., Summary: For clinical practice and research, these new findings offer new fundamental mechanisms for vascular calcification and provide new cautionary insights for therapeutic avenues.

Calcific diseases of the cardiovascular system, such as atherosclerotic calcification and calcific aortic valve disease, are widespread and clinically significant, causing substantial morbidity and mortality. Vascular cells, like bone cells, interact with their matrix substrate through molecular signals, and through biomechanical signals, such as traction forces transmitted from cytoskeleton to matrix. The interaction of contractile vascular cells with their matrix may be one of the most important factors controlling pathological mineralisation of the artery wall and cardiac valves. In many respects, the matricrine and matrix mechanical changes in calcific vasculopathy and valvulopathy resemble those occurring in embryonic bone development and normal bone mineralisation. The matrix proteins provide a microenvironment for propagation of crystal growth and provide mechanical cues to the cells that direct differentiation. Small contractions of the cytoskeleton may tug on integrin links to sites on matrix proteins, and thereby sense the stiffness, possibly through deformation of binding proteins causing release of differentiation factors such as products of the members of the transforming growth factor-β superfamily. Inflammation and matrix characteristics are intertwined: inflammation alters the matrix such as through matrix metalloproteinases, while matrix mechanical properties affect cellular sensitivity to inflammatory cytokines. The adhesive properties of the matrix also regulate self-organisation of vascular cells into patterns through reaction-diffusion phenomena and left-right chirality. In this review, we summarise the roles of extracellular matrix proteins and biomechanics in the development of inflammatory cardiovascular calcification., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.)

In calcific aortic valve disease, the valve cusps undergo retraction, stiffening, and nodular calcification. The inflammatory cytokine, tumor necrosis factor (TNF)-α, contributes to valve disease progression; however, the mechanisms of its actions on cusp retraction and stiffening are unclear. We investigated effects of TNF-α on murine aortic valvular interstitial cells (VICs) within three-dimensional, free-floating, compliant, collagen hydrogels, simulating their natural substrate and biomechanics. TNF-α increased retraction (percentage of diameter), stiffness, and formation of macroscopic, nodular structures with calcification in the VIC-laden hydrogels. The effects of TNF-α were attenuated by blebbistatin inhibition of myosin II-mediated cytoskeletal contraction. Inhibition of actin polymerization with cytochalasin-D, but not inhibition of Rho kinase with Y27632, blocked TNF-α-induced retraction in three-dimensional VIC hydrogels, suggesting that actin stress fibers mediate TNF-α-induced effects. In the hydrogels, inhibitors of NF-κB blocked TNF-α-induced retraction, whereas simultaneous inhibition of c-Jun N-terminal kinase was required to block TNF-α-induced stiffness. TNF-α also significantly increased collagen deposition, as visualized by Masson's trichrome staining, and up-regulated mRNA expression of discoidin domain receptor tyrosine kinase 2, fibronectin, and α-smooth muscle actin. In human aortic valves, calcified cusps were stiffer and had more collagen deposition than noncalcified cusps. These findings suggest that inflammation, through stimulation of cytoskeletal contractile activity, may be responsible for valvular cusp retraction, stiffening, and formation of calcified nodules., (Copyright © 2016 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Calcific aortic valve disease (CAVD) is increasingly prevalent worldwide with significant morbidity and mortality. Therapeutic options beyond surgical valve replacement are currently limited. In 2011, the National Heart Lung and Blood Institute assembled a working group on aortic stenosis. This group identified CAVD as an actively regulated disease process in need of further study. As a result, the Alliance of Investigators on CAVD was formed to coordinate and promote CAVD research, with the goals of identifying individuals at risk, developing new therapeutic approaches, and improving diagnostic methods. The group is composed of cardiologists, geneticists, imaging specialists, and basic science researchers. This report reviews the current status of CAVD research and treatment strategies with identification of areas in need of additional investigation for optimal management of this patient population., (© 2014 American Heart Association, Inc.)

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

Vascular calcification is recognized as an independent predictor of cardiovascular mortality, particularly in subjects with chronic kidney disease. However, the pathways by which dysregulation of lipid and mineral metabolism simultaneously occur in this particular population remain unclear. We have shown that activation of the farnesoid X receptor (FXR) blocks mineralization of bovine calcifying vascular cells (CVCs) and in ApoE knock-out mice with 5/6 nephrectomy. In contrast to FXR, this study showed that liver X receptor (LXR) activation by LXR agonists and adenovirus-mediated LXR overexpression by VP16-LXRα and VP16-LXRβ accelerated mineralization of CVCs. Conversely, LXR inhibition by dominant negative (DN) forms of LXRα and LXRβ reduced calcium content in CVCs. The regulation of mineralization by FXR and LXR agonists was highly correlated with changes in lipid accumulation, fatty acid synthesis, and the expression of sterol regulatory element binding protein-1 (SREBP-1). The rate of lipogenesis in CVCs through the SREBP-1c dependent pathway was reduced by FXR activation, but increased by LXR activation. SREBP-1c overexpression augmented mineralization in CVCs, whereas SREBP-1c DN inhibited alkaline phosphatase activity and mineralization induced by LXR agonists. LXR and SREBP-1c activations increased, whereas FXR activation decreased, saturated and monounsaturated fatty acids derived from lipogenesis. In addition, we found that stearate markedly promoted mineralization of CVCs as compared with other fatty acids. Furthermore, inhibition of either acetyl-CoA carboxylase or acyl-CoA synthetase reduced mineralization of CVCs, whereas inhibition of stearoyl-CoA desaturase induced mineralization. Therefore, a stearate metabolite derived from lipogenesis might be a risk factor for the development of vascular calcification.

Objective: Clinical and experimental studies demonstrate the important roles of vascular smooth muscle cells (VSMC) in the pathogenesis of atherosclerosis. We have previously determined that the osteogenic transcription factor Runx2 is essential for VSMC calcification. The present study characterized Runx2-regulated signals and their potential roles in vascular calcification., Methods and Results: In vivo studies with atherogenic apolipoprotein E(-/-) mice demonstrated that increased oxidative stress was associated with upregulation of Runx2 and receptor activator of nuclear factor κB ligand (RANKL), which colocalized in the calcified atherosclerotic lesions and were juxtaposed to infiltrated macrophages and osteoclast-like cells that are positively stained for an osteoclast marker, tartrate-resistant acid phosphatase. Mechanistic studies using RNA interference, a luciferase reporter system, chromatin immunoprecipitation, and electrophoretic mobility shift assays indicated that Runx2 regulated the expression of RANKL via a direct binding to the 5'-flanking region of the RANKL. Functional characterization revealed that RANKL did not induce VSMC calcification, nor was RANKL required for oxidative stress-induced VSMC calcification. Using a coculture system, we demonstrated that VSMC-expressed RANKL induced migration as well as differentiation of bone marrow-derived macrophages into multinucleated, tartrate-resistant acid phosphatase-positive osteoclast-like cells. These effects were inhibited by the RANKL antagonist osteoprotegerin and with VSMC deficient in Runx2 or RANKL., Conclusion: We demonstrate that Runx2 directly binds to the promoter and controls the expression of RANKL, which mediates the crosstalk between calcifying VSMC and migration and differentiation of macrophages into osteoclast-like cells in the atherosclerotic lesions. Our studies provide novel mechanistic insights into the regulation and function of VSMC-derived RANKL in the pathogenesis of atherosclerosis and vascular calcification.

Vascular calcification increasingly afflicts our aging, dysmetabolic population. Once considered only a passive process of dead and dying cells, data from multiple laboratories worldwide have converged to demonstrate that vascular calcification is a highly regulated form of biomineralization. The goal of this thematic review series is to highlight what is known concerning the biological "players" and "game rules" with respect to vascular mineral metabolism. Armed with this understanding, it is hoped that novel therapeutic strategies can be crafted to prevent and treat vascular calcium accrual, to the benefit of our patients afflicted with arteriosclerotic valvular and vascular diseases.

In the past decade, the prevalence, significance, and regulatory mechanisms of vascular calcification have gained increasing recognition. Over a century ago, pathologists recognized atherosclerotic calcification as a form of extraskeletal ossification. Studies are now identifying the mechanism of this remarkable process as a recapitulation of embryonic endochondral and membranous ossification through phenotypic plasticity of vascular cells that function as adult mesenchymal stem cells. These embryonic developmental programs, involving bone morphogenetic proteins and potent osteochondrogenic transcription factors, are triggered and modulated by a variety of inflammatory, metabolic, and genetic disorders, particularly hyperlipidemia, chronic kidney disease, diabetes, hyperparathyroidism, and osteoporosis. They are also triggered by loss of powerful inhibitors, such as fetuin A, matrix Gla protein, and pyrophosphate, which ordinarily restrict biomineralization to skeletal bone. Teleologically, soft-tissue calcification might serve to create a wall of bone to sequester noxious foci such as chronic infections, parasites, and foreign bodies. This Review focuses on atherosclerotic and medial calcification. The capacity of the vasculature to produce mineral in culture and to produce de novo, vascularized, trabecular bone and cartilage tissue, even in patients with osteoporosis, should intrigue investigators in tissue engineering and regenerative biology.

The role of atherosclerotic calcification in plaque rupture remains controversial. In previous analyses using finite element model analysis, circumferential stress was reduced by the inclusion of a calcium deposit in a representative human anatomical configuration. However, a recent report, also using finite element analysis, suggests that microscopic calcium deposits increase plaque stress. We used mathematical models to predict the effects of rigid and liquid inclusions (modeling a calcium deposit and a lipid necrotic core, respectively) in a distensible material (artery wall) on mechanical failure under uniaxial and biaxial loading in a range of configurations. Without inclusions, stress levels were low and uniform. In the analytical model, peak stresses were elevated at the edges of a rigid inclusion. In the finite element model, peak stresses were elevated at the edges of both inclusions, with minimal sensitivity to the wall distensibility and the size and shape of the inclusion. Presence of both a rigid and a soft inclusion enlarged the region of increased wall stress compared with either alone. In some configurations, the rigid inclusion reduced peak stress at the edge of the soft inclusion but simultaneously increased peak stress at the edge of the rigid inclusion and increased the size of the region affected. These findings suggest that the presence of a calcium deposit creates local increases in failure stress, and, depending on relative position to any neighboring lipid pools, it may increase peak stress and the plaque area at risk of mechanical failure.

Cardiovascular calcium deposition is associated with osteoporosis through various potential mechanisms involving molecular regulatory factors at the nanoscale level that govern skeletal bone and cardiovascular tissues. In this article, several possible mechanisms linking cardiovascular calcification and osteoporosis are discussed, including aging, tissue-specific responses to chronic inflammation, flow-limiting atherosclerosis of skeletal end arteries causing ischemic abnormalities in metabolism, shared endogenous regulatory factors that affect the two tissues in a reciprocal manner, and changes in a cysteine protease inhibitor, fetuin. Any or all of these factors and phenomena may contribute to the association.

We examined the effect of liver X receptor (LXR) agonists on vascular calcification, prevalent in atherosclerotic lesions. T0901317, an LXR agonist, augmented protein kinase A (PKA)-induced mineralization and alkaline phosphatase (ALP) activity in aortic smooth muscle cells isolated from wild-type, but not from Lxrbeta(-/-)mice. A six-hour T0901317 treatment augmented the PKA-induced expression of the phosphate transporter Pit-1, a positive regulator of mineralization, suggesting a direct role. A ten-day T0901317 treatment attenuated PKA-induced expression of mineralization inhibitors, osteopontin and ectonucleotide pyrophosphatase/phosphodiesterase-1, suggesting an indirect role. The effects of T0901317 were attenuated by inhibition of ALP, Pit-1 and Rho-associated kinase, but not by inhibition of PKA. These results suggest that T0901317-augmented mineralization occurs downstream of PKA, involving both direct and indirect LXR-mediated pathways.

Vascular calcification is associated with increased cardiovascular risk and occurs by osteochondrogenic differentiation of vascular cells. Many of the same regulatory factors that control skeletal mineralization, including the complex metabolic pathway controlling levels of the activator, inorganic phosphate, and the potent inhibitor, pyrophosphate, also govern vascular calcification. We previously found that the cAMP/PKA signaling pathway mediates in vitro vascular cell calcification induced by inflammatory factors including tumor necrosis factor-alpha 1 and oxidized phospholipids. In this report, we tested whether this signaling pathway modulates phosphate and pyrophosphate metabolism. Treatment of primary murine aortic cells with the PKA activator, forskolin, significantly induced osteoblastic differentiation markers, including alkaline phosphatase (ALP), osteopontin, and osteocalcin as well as the pyrophosphate generator, ectonucleotide-pyrophosphatase/phosphodiesterase-1 (Enpp1) and the pyrophosphate transporter, ankylosis protein, but not the sodium/phosphate cotransporter, Pit-1. In the presence of a substrate for ALP, beta-glycerophosphate, which generates inorganic phosphate, forskolin also enhanced matrix mineralization. Inhibitors of ALP or Pit-1 abrogated forskolin-induced osteopontin expression and mineralization but not forskolin-induced osteocalcin or ALP. These results suggest that phosphate is necessary for PKA-induced calcification of vascular cells and that the extent of PKA-induced calcification is controlled by feedback induction of the inhibitor, pyrophosphate.

Vascular calcification is widespread, particularly in patients with chronic kidney disease, who receive, among other treatments, active vitamin D supplements. Emerging evidence indicates that vascular calcification is a regulated process that resembles embryonic endochondral osteogenesis, involving osteoblastic differentiation of vascular smooth muscle cells. In experimental animal models, high dosages of vitamin D consistently promote vascular calcification. In particular, the vitamin D-fed rat is frequently used as a model to assess putative regulators of calcific vasculopathy. The artery wall calcification in these animals most likely results from multiple mechanisms involving systems physiology of the complex, bone-vascular-renal-endocrine axis. Genetically engineered mice with upregulated vitamin D signaling pathways have also shed light on the molecular intermediaries, including fibroblast growth factor-23 and transcriptional intermediary factor 1-alpha. In contrast to the studies of animals, studies of humans show that vitamin D has an inverse relationship or little effect. This difference between in vitro and in vivo findings is most likely, again, due to the complex, systemic feedback regulatory mechanisms that control calcium-phosphate metabolism. Recent epidemiologic evidence suggests that there is a narrow range of vitamin D levels in which vascular function is optimized. Levels above or below this range seem to confer a significant increase in risk for cardiovascular disease. There is some evidence to suggest that dietary vitamin D may be carried by lipoprotein particles into cells of the artery wall and atherosclerotic plaque, where it may be converted to active form by monocyte-macrophages. These findings raise interesting questions regarding the effects of vitamin D intake on atherosclerotic calcification and cardiovascular risk.

Vascular calcification is associated with increased cardiovascular morbidity and mortality and has long been associated with advanced atherosclerotic lesions. While vascular calcification is considered a surrogate marker for atherosclerosis, the mechanisms that link the two are poorly understood. The consensus of recent research is that active regulatory processes govern vascular calcification, and much focus has been placed on elucidating the phenomenon of atherosclerotic calcification. Building upon extensive in vitro work and the previous development of atherosclerotic murine models, several groups have developed murine models of atherosclerotic calcification. From imposing chronic renal failure to developing double-knockout mice, this recent work has provided insight into the pathophysiology of mineralized matrix formation in atherosclerotic lesions, as well as development of potential therapies to prevent or inhibit progression of calcified plaque. The aim is to briefly review current understanding of the molecular basis for atherosclerotic calcification and to discuss some murine models that may be useful in advancing knowledge of its mechanisms.

Background: The role of osteoprotegerin in vascular disease is unclear. Recent observational studies show that serum osteoprotegerin levels are associated with the severity and progression of coronary artery disease, atherosclerosis, and vascular calcification in patients. However, genetic and treatment studies in mice suggest that osteoprotegerin may protect against vascular calcification., Methods and Results: To test whether osteoprotegerin induces or prevents vascular disease, we treated atherogenic diet-fed ldlr(-/-) mice with recombinant osteoprotegerin (Fc-OPG) or vehicle for 5 months. Vehicle-treated mice developed significant, progressive atherosclerosis with increased plasma osteoprotegerin levels, consistent with observational studies, and approximately 15% of these atherosclerotic lesions developed calcified cartilage-like metaplasia. Treatment with Fc-OPG significantly reduced the calcified lesion area without affecting atherosclerotic lesion size or number, vascular cytokines, or plasma cholesterol levels. Treatment also significantly reduced tissue levels of aortic osteocalcin, a marker of mineralization., Conclusions: These data support a role for osteoprotegerin in the vasculature as an inhibitor of calcification and a marker, rather than a mediator, of atherosclerosis.

Objective: Clinical studies demonstrate that mineralocorticoid receptor (MR) antagonism improves outcomes in cardiovascular patients and that vascular calcification correlates with adverse cardiac events. We have recently demonstrated that human vascular smooth muscle cells (VSMCs) express functional MRs that, in response to aldosterone, modulate expression of osteogenic genes including alkaline phosphatase (ALP) and bone morphogenetic protein-2 (BMP2). This study examines the effects of MR activation by aldosterone on the process of in vitro vascular calcification., Methods and Results: Using immunoblotting and adenoviral promoter-reporter assays, we demonstrated that calcifying vascular cells (CVCs), an in vitro model of vascular calcification, express MRs that mediate both aldosterone- and cortisol-stimulated gene transcription. In this model, aldosterone stimulated ALP activity, an early marker of osteoblastic differentiation, as well as mineralization. Aldosterone antagonism with spironolactone abolished both effects implicating CVC MRs in the mechanism of aldosterone-stimulated vascular calcification. Inhibition of BMP2 signaling by overexpression of dominant negative BMP2 receptor did not attenuate aldosterone-induced osteoblastic differentiation., Conclusions: Aldosterone activation of MR promotes osteoblastic differentiation and mineralization of VSMCs independent of BMP2 signaling. These data provide a mechanistic link between hormone-mediated VSMC MR activation and vascular calcification, two processes associated with increased risk of cardiovascular ischemic events in humans.

Vascular calcification is associated with atherosclerosis, but whether it mechanically affects plaque stability remains controversial. To assess the effect of mineralization on plaque vulnerability to mechanical shear stress, we applied fluid shear to cultures of calcifying vascular cells (CVC), a subpopulation of smooth muscle cells that spontaneously mineralize. CVC cultures containing nodules were treated for 10 days with vehicle control or beta-glycerophosphate (BGP) to accelerate mineralization. Cultures were placed in a parallel-plate flow system and were subjected to increasing fluid shear stress (4.9 dyn/cm(2)/min up to 400 dyn/cm(2)). The number of nodules remaining attached was recorded every 10 min. Results showed that control cultures and BGP-treated cultures, which contained significantly greater calcium mineral than control cultures, had similar detachment thresholds (50-100 dyn/cm(2)), with linear portions of their stress/detachment curves from 100 to 275 dyn/cm(2). Based on repeated measure analysis of variance, BGP-treated nodules were no more likely to detach at a given shear than controls, although they showed a trend toward greater stability. Thus, calcification does not appear to increase plaque vulnerability to fluid shear stress, although it may contribute to a slight stabilization. This model may represent the first in vitro model of mechanical rupture of atherosclerotic plaque.

Vascular calcification develops within atherosclerotic lesions and results from a process similar to osteogenesis. One of the paracrine regulators of bone-derived osteoblasts, insulin-like growth factor-I (IGF-I), is also present in atherosclerotic lesions. To evaluate its possible role in vascular calcification, we assessed its in vitro effects on proliferation and differentiation in calcifying vascular cells (CVCs), a subpopulation of bovine aortic medial cells. Results showed that IGF-I inhibited spontaneous CVC differentiation and mineralization as evidenced by decreased alkaline phosphatase (AP) activity and decreased matrix calcium incorporation, respectively. Furthermore, IGF-I inhibited the AP activity induced by bacterial lipopolysaccharide, TNF-alpha, or H2O2. It also induced CVC proliferation based on 3H-thymidine incorporation. Results from Northern analysis and tests using IGF-I analogs suggest that IGF-I effects are mediated through the IGF-I receptor. IGF-I also activated both the extracellular signal-regulated protein kinase (ERK) and phosphatidylinositol 3-kinase (PI3K) pathways. Inhibition of either the ERK or PI3K pathway reversed IGF-I effects on CVC proliferation and AP activity, suggesting a common downstream target. Overexpression of ERK activator also mimicked IGF-I inhibition of lipopolysaccharide-induced AP activity. These results suggest that IGF-I promotes proliferation and inhibits osteoblastic differentiation and mineralization of vascular cells via both ERK and PI3K pathways.

Vascular calcification, long thought to result from passive degeneration, involves a complex, regulated process of biomineralization resembling osteogenesis. Evidence indicates that proteins controlling bone mineralization are also involved in the regulation of vascular calcification. Artery wall cells grown in culture are induced to become osteogenic by inflammatory and atherogenic stimuli. Furthermore, osteoclast-like cells are found in calcified atherosclerotic plaques, and active resorption of ectopic vascular calcification has been demonstrated. In general, soft tissue calcification arises in areas of chronic inflammation, possibly functioning as a barrier limiting the spread of the inflammatory stimulus. Atherosclerotic calcification may be one example of this process, in which oxidized lipids are the inflammatory stimulus. Calcification is widely used as a clinical indicator of atherosclerosis. It progresses nonlinearly with time, following a sigmoid-shaped curve. The relationship between calcification and clinical events likely relates to mechanical instability introduced by calcified plaque at its interface with softer, noncalcified plaque. In general, as calcification proceeds, interface surface area increases initially, but eventually decreases as plaques coalesce. This phenomenon may account for reports of less calcification in unstable plaque. Vascular calcification is exacerbated in certain clinical entities, including diabetes, menopause, and osteoporosis. Mechanisms linking them must be considered in clinical decisions. For example, treatments for osteoporosis may have unanticipated effects on vascular calcification; the converse also applies. Further understanding of processes governing vascular calcification may yield new therapeutic options for vascular disease.

Purpose of Review: Vascular calcification occurs more often and earlier in patients with end-stage renal disease than in normal controls. It is a regulated biological process following many of the cellular and molecular programs in osteogenesis. This review summarizes some of the regulatory mechanisms that may explain its severity in renal patients., Recent Findings: A subpopulation of cells from arteries and cardiac valves produce a mineralizing matrix and undergo osteoblastic differentiation. Osteogenic differentiation regulators are found in calcified but not normal arteries. Phosphate levels have dramatic effects on vascular calcification in vitro, through a sodium phosphate transporter signaling molecular changes. Atherogenic oxidized lipids promote osteoblastic differentiation of vascular cells and inhibit bone mineralization. In uremic patients, the severity of dyslipidemia corresponds with the progression of vascular calcification. Oxidative stress and inflammatory mediators may underlie the effects of oxidized lipids. In dialysis patients, the degree of cardiac valvular calcification corresponds with levels of C-reactive protein. Genetic factors may also contribute. Polymorphisms of the inflammatory adhesion molecule, E-selectin, associate with coronary calcification in young women. Mice deficient in matrix GLA protein, which inhibits bone morphogenetic protein activity, develop complete ossification of the aorta, presumably as a result of unopposed osteogenic activity on vascular mesenchyme. Since matrix GLA protein function requires gamma-carboxylation of its glutamate residues by a vitamin K dependent carboxylase, warfarin treatment may affect vascular calcification by blocking vitamin K and hence matrix GLA protein activity., Summary: These findings indicate that vascular calcification is regulated both positively and negatively by a wide variety of mechanisms affecting patients with renal disease.

Background: Calcification is a common complication of atherosclerosis and other chronic inflammatory processes that involves infiltration of monocytes and accumulation of macrophages., Methods and Results: To determine whether these cells modulate vascular calcification in vitro, calcifying vascular cells (CVCs), a subpopulation of osteoblast-like cells derived from the artery wall, were cocultured with human peripheral blood monocytes for 5 days. Results showed that alkaline phosphatase (ALP) activity, a marker of osteoblastic differentiation, was significantly greater in cocultures than in cultures of CVCs or monocytes alone. Both ALP activity and matrix mineralization increased in proportion to the number of monocytes added. Activation of monocyte/macrophages (M/Ms) by oxidized LDL further increased ALP activity in cocultures. However, neither conditioned medium from oxidized-LDL-activated M/Ms or transwell coculture had this effect on CVCs, which suggests a need for cell-to-cell contact. In contrast, conditioned medium from lipopolysaccharide-activated M/Ms increased ALP activity of CVCs. ELISA showed that lipopolysaccharide-activated M/Ms secreted tumor necrosis factor-alpha, and neutralizing antibody to tumor necrosis factor-alpha attenuated the induction of ALP activity by the conditioned media., Conclusions: These results suggest that M/Ms enhance in vitro vascular calcification via 2 independent mechanisms: cell-cell interaction and production of soluble factors such as tumor necrosis factor-alpha.

BackgroundFibroblast growth factor 23 (FGF23) levels are elevated in chronic kidney disease (CKD) and elevated values have been associated with both heart disease and mortality. Recent studies show that FGF23, a protein synthesized by osteocytes, is also present in calcified atherosclerotic plaques and may be induced by heart disease. Whether vascular expression of FGF23 is associated with progressive CKD, however, remains unknown. Therefore, the relationship between kidney function, vascular calcification and FGF23 expression was evaluated in patients with heart disease.MethodsImmunohistochemistry for FGF23 was performed in coronary arteries of all patients undergoing heart transplantation at UCLA between February 2008 and 2010. Immunohistochemical staining for Klotho, DMP1, FGFR1, and FGFR3; calcium deposition; and RNA expression of Klotho and DMP1 were assessed in a subset of eight samples.ResultsFGF23 was detected by immunohistochemistry in 56% of the coronary artery specimens. Vascular FGF23 expression correlated with declining kidney function, as evidenced by reduced creatinine clearance. FGFR1 and FGFR3 were detected throughout the vascular tissue and in calcified plaques. Calcium deposition, Klotho expression and DMP1 expression correlated with FGF23 immunoreactivity.ConclusionsThe findings suggest that the Klotho-FGF23-FGFR system is active in coronary arteries and its upregulation correlates with impaired renal function and matrix calcium deposition.

The role of atherosclerotic calcification in plaque rupture remains controversial. In previous analyses using finite element model analysis, circumferential stress was reduced by the inclusion of a calcium deposit in a representative human anatomical configuration. However, a recent report, also using finite element analysis, suggests that microscopic calcium deposits increase plaque stress. We used mathematical models to predict the effects of rigid and liquid inclusions (modeling a calcium deposit and a lipid necrotic core, respectively) in a distensible material (artery wall) on mechanical failure under uniaxial and biaxial loading in a range of configurations. Without inclusions, stress levels were low and uniform. In the analytical model, peak stresses were elevated at the edges of a rigid inclusion. In the finite element model, peak stresses were elevated at the edges of both inclusions, with minimal sensitivity to the wall distensibility and the size and shape of the inclusion. Presence of both a rigid and a soft inclusion enlarged the region of increased wall stress compared with either alone. In some configurations, the rigid inclusion reduced peak stress at the edge of the soft inclusion but simultaneously increased peak stress at the edge of the rigid inclusion and increased the size of the region affected. These findings suggest that the presence of a calcium deposit creates local increases in failure stress, and, depending on relative position to any neighboring lipid pools, it may increase peak stress and the plaque area at risk of mechanical failure.