Vascular Calcification In Atherosclerosis Research Articles

Sortilin Is Upregulated in Osteoarthritis-Dependent Cartilage Calcification and Associated with Cellular Senescence.

Osteoarthritis (OA) is a chronic joint disease characterized by articular cartilage calcification, loss of articular cartilage, bone changes, pain, and disability. Cartilage calcification is one hallmark of OA and is predominantly caused by basic calcium crystals formed due to an imbalance of the pyrophosphate pathway. Sortilin is a transmembrane protein that contributes to vascular calcification in atherosclerosis by externalizing alkaline phosphatase (ALP)-containing vesicles. Calcification in atherosclerosis and osteoarthritis has been associated with cellular senescence. The aim of this study was to investigate the potential role of sortilin and senescence in osteoarthritis-dependent cartilage calcification. Osteoarthritic cartilage from human knee joints was collected after joint replacement, and samples were analyzed by immunohistochemistry and quantitative RT-PCR analysis. Human chondrocytes were treated with osteogenic medium for up to 21 days to induce calcification. Western blots for sortilin and ALP, as well as an ALP activity assay, were performed. Human chondrocytes were treated with mitomycin C to induce senescence, and sortilin expression was quantified at the protein and gene levels. Sections of knee joints from a murine model of osteoarthritis were stained for sortilin and p16 and analyzed by immunohistochemistry. Treatment of wild-type chondrocytes using an osteogenic medium similar to human chondrocytes was performed. Osteoarthritic cartilage from mouse and human knee joints showed an increased number of sortilin and p16-positive chondrocytes compared to healthy cartilage. This observation was corroborated by increased gene expression of sortilin and p16 in mild and moderate osteoarthritic cartilage samples. To investigate the mechanism of sortilin regulation, human chondrocytes were treated with osteogenic medium to induce calcification. Sortilin protein levels and expression were increased after 7 days of stimulation, whereas ALP levels and activity were upregulated after 21 days of stimulation. Similar observations were made in a murine osteoarthritis model. Mechanistically, senescent chondrocytes induced by mitomycin C showed an upregulation of sortilin and ALP gene expression compared to non-senescent chondrocytes. Our data indicate that sortilin and ALP are upregulated during cartilage calcification, which is associated with chondrocyte senescence and thus might contribute to the pathogenesis of osteoarthritis. Cellular senescence seems to induce sortilin expression.

Pathological Role of Receptor for Advanced Glycation End Products in Calcified Aortic Valve Stenosis.

BackgroundAortic stenosis (AS) is highly prevalent in patients with atherosclerotic cardiovascular disease. Advanced glycation end products (AGEs) and the receptor for AGEs (RAGE) play a pivotal role for vascular calcification in atherosclerosis. We hypothesize that the AGEs–RAGE axis could also be involved in the pathophysiological mechanism of calcified AS.Methods and ResultsA total of 54 patients with calcified AS who underwent aortic valve replacement were prospectively enrolled from 2014 to 2016 (mean age 75.3±7.7 years). Aortic valve specimens were obtained from 47 patients and 16 deceased control subjects without aortic valve disease (mean age 63.2±14.5 years). The valvular expression of RAGE was evaluated by immunohistochemistry. Serum levels of AGEs and soluble RAGE were measured in 50 patients with calcified AS and 70 age‐matched and sex‐matched control subjects without heart disease. The valvular RAGE expression in patients with calcified AS was higher than controls (P=0.004) and was significantly associated with a decreased ankle‐brachial pressure index (P=0.007) and an increased intima‐media thickness (P=0.026). RAGE and α–smooth muscle actin were coexpressed and were partially costained with osteocalcin and alkaline phosphatase. The serum levels of AGEs and soluble RAGE were significantly higher in the patients with calcified AS than in the controls (P=0.013 and P<0.001, respectively). Soluble RAGE (inversely) and use of aspirin were independently correlated with changes in left ventricular systolic function after aortic valve replacement (P=0.012 and P=0.002, respectively).ConclusionsOur present study suggests that RAGE may play a role in the pathogenesis of calcified AS, which is a prognostic marker in patients with AS after aortic valve replacement.

A Role for MMP-10 (Matrix Metalloproteinase-10) in Calcific Aortic Valve Stenosis.

Aortic valve (AV) calcification plays an important role in the progression of aortic stenosis (AS). MMP-10 (matrix metalloproteinase-10 or stromelysin-2) is involved in vascular calcification in atherosclerosis. We hypothesize that MMP-10 may play a pathophysiological role in calcific AS. Approach and Results: Blood samples (n=112 AS and n=349 controls) and AVs (n=88) from patients undergoing valve replacement were analyzed. Circulating MMP-10 was higher in patients with AS compared with controls (P<0.001) and correlated with TNFα (tumor necrosis factor α; rS=0.451; P<0.0001). MMP-10 was detected by immunochemistry in AVs from patients with AS colocalized with aortic valve interstitial cells markers α-SMA (α-smooth muscle actin) and vimentin and with calcification markers Runx2 (Runt-related transcription factor 2) and SRY (sex-determining region Y)-box 9. MMP-10 expression in AVs was further confirmed by RT-qPCR and western blot. Ex vivo, MMP-10 was elevated in the conditioned media of AVs from patients with AS and associated with interleukin-1β (rS=0.5045, P<0.001) and BMP (bone morphogenetic protein)-2 (rS=0.5003, P<0.01). In vitro, recombinant human MMP-10 induced the overexpression of inflammatory, fibrotic, and osteogenic markers (interleukin-1β, α-SMA, vimentin, collagen, BMP-4, Sox9, OPN [osteopontin], BMP-9, and Smad 1/5/8; P<0.05) and cell mineralization in aortic valve interstitial cells isolated from human AVs, in a mechanism involving Akt (protein kinase B) phosphorylation. These effects were prevented by TIMP-1 (tissue inhibitor of metalloproteinases type 1), a physiological MMP inhibitor, or specifically by an anti-MMP-10 antibody. MMP-10, which is overexpressed in aortic valve from patients with AS, seems to play a central role in calcification in AS through Akt phosphorylation. MMP-10 could be a new therapeutic target for delaying the progression of aortic valve calcification in AS.

Hydroxyapatite-binding micelles for the detection of vascular calcification in atherosclerosis.

Atherosclerosis is a chronic disease characterized by the formation of calcified, arterial plaques. Microcalcifications (5 μm to 100 μm), mainly composed of hydroxyapatite (HA, Ca5(PO4)3(OH)), develop in the fibrous caps of atherosclerotic plaques and can trigger plaque rupture due to the loss of compliance and elasticity. Ultimately, plaque rupture can cause arterial occlusion and embolization and result in ischemic events such as strokes and myocardial infarctions. Unfortunately, current imaging technologies used to detect calcifications are limited by low signal-to-noise ratio or use invasive procedures that pose risk of arterial dissection. To mitigate these drawbacks, in our study, we developed a novel, fluorescently-labeled peptide amphiphile micelle (PAM) that uses a 12 amino acid HA-binding peptide (HABP) [SVSVGMKPSPRP] to target and detect atherosclerotic calcification (HA PAM). Our results show HA PAMs can successfully target HA microcrystals with a strong binding affinity (KD = 6.26 ± 1.2 μM) in vitro. In addition, HA PAMs detected HA mineralization (HA PAM vs. non-targeting micelle, p≤ 0.001; HA PAM vs. scrambled HABP PAM, p≤ 0.01) formed by calcifying mouse aortic vascular smooth muscle cells (MOVAS). Moreover, HA PAMs successfully detected calcifications in atherosclerotic mouse models as well as in patient-derived arteries. Our studies show that HA PAMs show promise as calcium-targeting nanoparticles for the detection of calcifications in atherosclerosis.

Diabetic Vascular Calcification Mediated by the Collagen Receptor Discoidin Domain Receptor 1 via the Phosphoinositide 3-Kinase/Akt/Runt-Related Transcription Factor 2 Signaling Axis.

Objective- Vascular calcification is a common and severe complication in patients with atherosclerosis which is exacerbated by type 2 diabetes mellitus. Our laboratory recently reported that the collagen receptor discoidin domain receptor 1 (DDR1) mediates vascular calcification in atherosclerosis; however, the underlying mechanisms are unknown. During calcification, vascular smooth muscle cells transdifferentiate into osteoblast-like cells, in a process driven by the transcription factor RUNX2 (runt-related transcription factor 2). DDR1 signals via the phosphoinositide 3-kinase/Akt pathway, which is also central to insulin signaling, and upstream of RUNX2, and this led us to investigate whether DDR1 promotes vascular calcification in diabetes mellitus via this pathway. Approach and Results- Ddr1+/+ ; Ldlr-/- (single knock-out) and Ddr1-/- ; Ldlr-/- (double knock-out) mice were placed on high-fat diet for 12 weeks to induce atherosclerosis and type 2 diabetes mellitus. Von Kossa staining revealed reduced vascular calcification in the aortic arch of double knock-out compared with single knock-out mice. Immunofluorescent staining for RUNX2 was present in calcified plaques of single knock-out but not double knock-out mice. Primary vascular smooth muscle cells obtained from Ddr1+/+ and Ddr1-/- mice were cultured in calcifying media. DDR1 deletion resulted in reduced calcification, a 74% reduction in p-Akt levels, and an 88% reduction in RUNX2 activity. Subcellular fractionation revealed a 77% reduction in nuclear RUNX2 levels in Ddr1-/- vascular smooth muscle cells. DDR1 associated with phosphoinositide 3-kinase, and treatment with the inhibitor wortmannin attenuated calcification. Finally, we show that DDR1 is important to maintain the microtubule cytoskeleton which is required for the nuclear localization of RUNX2. Conclusions- These novel findings demonstrate that DDR1 promotes RUNX2 activity and atherosclerotic vascular calcification in diabetes mellitus via phosphoinositide 3-kinase/Akt signaling.

Vascular Calcification: Is it rather a Stem/Progenitor Cells Driven Phenomenon?

Vascular calcification (VC) has witnessed a surge of interest. Vasculature is virtually an omnipresent organ and has a notably high capacity for repair throughout embryonic and adult life. Of the vascular diseases, atherosclerosis is a leading cause of morbidity and mortality on account of ectopic cartilage and bone formation. Despite the identification of a number of risk factors, all the current theories explaining pathogenesis of VC in atherosclerosis are far from complete. The most widely accepted response to injury theory and smooth muscle transdifferentiation to explain the VC observed in atherosclerosis is being challenged. Recent focus on circulating and resident progenitor cells in the vasculature and their role in atherogenesis and VC has been the driving force behind this review. This review discusses intrinsic cellular players contributing to fate determination of cells and tissues to form ectopic cartilage and bone formation.

Abstract 383: STIM1 Deficiency in Vascular Smooth Muscle Cells Promotes Vascular Calcification in Atherosclerosis

Vascular calcification is a characteristic feature of atherosclerosis. We and others have demonstrated that osteogenic differentiation of vascular smooth muscle cells (VSMC) contribute predominantly to the pathogenesis of vascular calcification in atherosclerosis. The key cellular event that leads to calcification is the secretion of matrix vesicles (MVs). However, the molecular regulation of MV release and the causal effect of MV release on VSMC calcification are poorly understood. The objective of this study is to investigate the function of a key calcium flux regulator, stromal interaction molecule 1 (STIM1), in regulating MV release and VSMC calcification; and to elucidate the underlying molecular mechanisms. SMC-specific STIM1-deficient mice (STIM1 Δ/Δ SMC ) were generated by breeding SM22α-Cre mice with STIM1 smooth muscle floxed mice (STIM1 f/f ). In vitro characterization using primary VSMC isolated from STIM1 Δ/Δ SMC and the control STIM1 f/f mice demonstrated that STIM1 deletion promoted VSMC calcification, although STIM1 deficiency has been linked to decreased calcium signals in smooth muscle cells. Increased release of MVs was demonstrated with the STIM1 Δ/Δ SMC VSMC compared with the control STIM1 f/f VSMC. Using the atherogenic ApoE -/- model, we demonstrated that SMC-specific STIM1 deficiency increased atherosclerotic vascular calcification in vivo. Consistently, increased MVs were determined in the serum of the STIM1 Δ/Δ SMC mice. Mechanistically, we found that STIM1 deficiency did not affect VSMC proliferation and survival, but decreased the expression of SMC-specific α-actin. The size of MVs released from STIM1 f/f and STIM1 Δ/Δ SMC VSMC appeared similar. However, MVs from the STIM1 Δ/Δ SMC VSMC contained a greater amount of calcium compared with those from the STIM1 f/f VSMC. Furthermore, immunofluorescent staining identified a rearrangement of actin-filament structure in the STIM1 Δ/Δ VSMC, a critical cellular event that controls the release of MVs. These studies have determined a new causative effect of VSMC-expressed STIM1 on atherosclerotic vascular calcification; and identified a novel link connecting STIM1 and actin-cytoskeleton rearrangement in regulating MV release and vascular calcification.

Abstract 241: O-GlcNAcylation of Runx2 in Nuclear Matrix Targeting Signal Modulates Vascular Calcification

Vascular calcification is prevalent in patients with atherosclerosis, diabetes, and chronic kidney diseases and increases the mortality and morbidity of those patients. Osteogenic differentiation of vascular smooth muscle cells (VSMC) has been recognized as a key feature of the calcification process. Using smooth muscle-specific deletion mouse model, we have demonstrated an essential role of SMC-derived Runx2 in regulating vascular calcification in atherosclerosis in vivo. The present study further defined Runx2 regulation and its functional domains that control osteogenesis of VSMC. Using a serial of Runx2 truncation mutants, we located the domain between amino acids 391 and 432 as responsible region for Runx2 osteogenic function in VSMC. This region contains the nuclear matrix targeting signal, which has been shown to interact with Smad proteins. Accordingly, we determined the contribution of Smad proteins in Runx2-regulated VSMC calcification. By knockdown individual Smad in VSMC, we demonstrated that Smads 1/5/8, but not Smads 2/3, were required for Runx2 osteogenic function and VSMC calcification. In addition, we found the osteogenic function of Runx2 was abolished by inhibition of protein O-GlcNAcylation, a unique posttranslational modification that we have recently reported to be critical in regulating VSMC calcification. Runx2 O-GlcNAc modification was further demonstrated by immunoprecipitation. Based on bioinformatics analysis, we found several putative O-GlcNAcylation sites within Runx2 osteogenic domain. Using site-directed mutagenesis, we demonstrated that mutation on S385, S387, T404, T406, T412, S413, or T427 decreased Runx2 O-GlcNAcylation and Runx2 binding to Smads 1/5/8, and thus decreasing Runx2 activity and VSMC calcification. In summary, we have identified the Runx2 osteogenic functional domain in VSMC; and demonstrated that O-GlcNAc modification of Runx2 in its osteogenic functional domain is critical for Runx2-regualted VSMC calcification. Our studies provide molecular insights into the regulation and function of Runx2 in VSMC calcification, which may shed lights on novel targets that are amenable to drug discovery for vascular calcification.

Differentiation of Vascular Stem Cells Contributes to Ectopic Calcification of Atherosclerotic Plaque.

The cellular and molecular basis of vascular calcification (VC) in atherosclerosis is not fully understood. Here, we investigate role of resident/circulating progenitor cells in VC and contribution of inflammatory plaque environment to this process. Vessel-derived stem/progenitor cells (VSCs) and mesenchymal stem cells (MSCs) isolated from atherosclerotic ApoE(-/-) mice showed significantly more in vitro osteogenesis and chondrogenesis than cells generated from control C57BL/6 mice. To assess their ability to form bone in vivo, cells were primed chondrogenically or cultured in control medium on collagen glycosaminoglycan scaffolds in vitro prior to subcutaneous implantation in ApoE(-/-) and C57BL/6 mice using a crossover study design. Atherosclerotic ApoE(-/-) MSCs and VSCs formed bone when implanted in C57BL/6 mice. In ApoE(-/-) mice, these cells generated more mature bone than C57BL/6 cells. The atherosclerotic in vivo environment alone promoted bone formation by implanted C57BL/6 cells. Un-primed C57BL/6 VSCs were unable to form bone in either mouse strain. Treatment of ApoE(-/-) VSC chondrogenic cultures with interleukin (IL)-6 resulted in significantly increased glycosaminoglycan deposition and expression of characteristic chondrogenic genes at 21 days. In conclusion, resident vascular cells from atherosclerotic environment respond to the inflammatory milieu and undergo calcification. IL-6 may have a role in aberrant differentiation of VSCs contributing to vascular calcification in atherosclerosis.

Can the NK Family of Osteoblast Homeodomain Transcription Factors Signaling Be a Magic Bullet to Reverse Calcification-Induced Vasculopathy in Diabetes?

Human vascular calcification burden has existed for at least 5 millennia and has long been a major area of interest in the cardiovascular physiopathology research. Abnormal calcium deposition occurs in almost all arterial beds in both the media and intima in metabolic and diabetic diseases. Calcification of arteries reduces arterial elastance and compromises cardiovascular hemodynamics, which are high risk factors for cardiovascular diseases such stroke and ischemic heart disease (1). Despite the wealth of information regarding the effects of vascular calcification in atherosclerosis, metabolic syndrome, hypertension, and diabetes, the molecular mechanism of arteriosclerosis remains poorly understood. Therefore, understanding the mechanism by which diabetes and metabolic disease induce vascular calcification is crucial to identify effective new therapeutic strategies to intervene with the disease. Interestingly in this issue, Cheng et al. (2) determined the in vivo contribution of members of the highly conserved NK family of osteoblast homeodomain transcription factors 1 ( Msx1 ) and 2 ( Msx2 ) signaling in arteriosclerosis and vascular stiffness in diabetic mice. The study by Cheng et al. provides new insight into the molecular mechanism that drives arteriosclerosis in diabetes and suggests a potential target for future therapy. Thus, targeting Msx1 …

Urokinase receptor mediates osteoclastogenesis via M-CSF release from osteoblasts and the c-Fms/PI3K/Akt/NF-κB pathway in osteoclasts.

Bone remodeling is a dynamic process based on a fine-tuned balance between formation and degradation of bone. Osteoblasts (OBLs) are responsible for bone formation and bone resorption is mediated by osteoclasts (OCLs). The mechanisms regulating the OBL-OCL balance are critical in health and disease; however, they are still far from being understood. We reported recently that the multifunctional urokinase receptor (uPAR) mediates osteogenic differentiation of mesenchymal stem cells (MSCs) to OBLs and vascular calcification in atherosclerosis. Here, we address the question of whether uPAR may also be engaged in regulation of osteoclastogenesis. We show that uPAR mediates this process in a dual fashion. Thus, uPAR affected OBL-OCL interplay. We observed that osteoclastogenesis was significantly impaired in co-culture of monocyte-derived OCLs and in OBLs derived from MSCs lacking uPAR. We show that expression and release, from OBLs, of macrophage colony-stimulating factor (M-CSF), which is indispensable for OCL differentiation, was inhibited by uPAR loss. We further found that uPAR, on the other hand, controlled formation, differentiation, and functional properties of macrophage-derived OCLs. Expression of osteoclastogenic markers, such as tartrate-resistant acid phosphatase (TRAP) and cathepsin K, was impaired in OCLs derived from uPAR-deficient macrophages. The requirement of uPAR for osteoclastogenesis was further confirmed by immunocytochemistry and in bone resorption assay. We provide evidence that the underlying signaling mechanisms involve uPAR association with the M-CSF binding receptor c-Fms followed by c-Fms phosphorylation and activation of the PI3K/Akt/NF-κB pathway in OCLs. We further show that uPAR uses this pathway to regulate a balance between OCL differentiation, apoptosis, and cell proliferation. Our study identified uPAR as an important and multifaceted regulator of OBL-OCL molecular interplay that may serve as an attractive target in bone disease and ectopic calcification.

Necrotic and apoptotic cells serve as nuclei for calcification on osteoblastic differentiation of human mesenchymal stem cells in vitro

A close relationship between cell death and pathological calcification has recently been reported, such as vascular calcification in atherosclerosis. However, the roles of cell death in calcification by osteoblast lineage have not been elucidated in detail. In this study, we investigated whether cell death is involved in the calcification on osteoblastic differentiation of human bone marrow mesenchymal stem cells (hMSC) under osteogenic culture in vitro. Apoptosis and necrosis occurred in an osteogenic culture of hMSC, and cell death preceded calcification. The generation of intracellular reactive oxygen species, chromatin condensation and fragmentation, and caspase-3 activation increased in this culture. A pan-caspase inhibitor (Z-VAD-FMK) and anti-oxidants (Tiron and n-acetylcysteine) inhibited osteogenic culture-induced cell death and calcification. Furthermore, calcification was significantly promoted by the addition of necrotic dead cells or its membrane fraction. Spontaneously dead cells by osteogenic culture and exogenously added necrotic cells were surrounded by calcium deposits. Induction of localized cell death by photodynamic treatment in the osteogenic culture resulted in co-localized calcification. These findings show that necrotic and apoptotic cell deaths were induced in an osteogenic culture of hMSC and indicated that both necrotic and apoptotic cells of osteoblast lineage served as nuclei for calcification on osteoblastic differentiation of hMSC in vitro.

Cross-Talk of Receptor Activator of Nuclear Factor-κB Ligand Signaling With Renin–Angiotensin System in Vascular Calcification

Vascular calcification is accelerated by hypertension and also contributes to hypertension; however, it is an enigma why hypertension and vascular calcification are a vicious spiral. The present study elucidates the cross-talk between renin-angiotensin II system and receptor activator of nuclear factor-κB ligand (RANKL) system in vascular calcification. Angiotensin (Ang) II (10(-7) mol/L) significantly increased calcium deposition as assessed by Alizarin Red staining, associated with a significant increase in the expression of RANKL, RANK, and bone-related genes, such as cbfa1 and msx2, in human aortic vascular smooth muscle cells. Infusion of Ang II (100 ng/kg per minute) in ovariectomized ApoE(-/-) mice under high-fat diet significantly increased the expression of RANKL system and calcification in vivo, whereas administration of Ang II receptor blocker (olmesartan, 3 mg/kg per day) decreased the calcification and bone markers' expression. In addition, male OPG(-/-) mice showed a significant increase in vascular calcification followed by Ang II infusion as compared with wild type. Conversely, RANKL significantly increased Ang II type 1 receptor and angiotensin II-converting enzyme expression in vascular smooth muscle cells via extracellular signal-regulated protein kinase phosphorylation. The present study demonstrated that Ang II significantly induced vascular calcification in vitro and in vivo through RANKL activation. In addition, RANKL activated renin-angiotensin II system, especially angiotensin II-converting enzyme and Ang II type 1 receptor. Cross-talk between renin-angiotensin II system and RANKL system might work as a vicious cycle to promote vascular calcification in atherosclerosis. Further studies to inhibit renin-angiotensin II system and RANKL may provide new therapeutic options to prevent and regress vascular calcification.

Proinflammatory Vascular Calcification

See related article, pages 1248–1256 Vascular calcification occurs as part of atherosclerosis. It is caused by the deposition of basic calcium phosphate (BCP) crystals in what appears to be a highly regulated process resembling bone formation.1–4 Vascular calcification occurs in several forms,1–4 including small crystal deposits in the neointima and large calcified areas in more advanced atherosclerotic plaques, which frequently take on the appearance of ectopic bone. Media sclerosis (Moncke-berg disease) is a form of calcification associated with the elastic lamellae and is distinct from atherosclerotic calcification.1,3,4 It is common in diabetes mellitus and renal failure and is often seen in combination with atherosclerosis. Studies on the mechanism of vascular calcification have in most cases focused on similarities to bone formation1–3; however, there are likely to be unique characteristics in the regulation of each type of calcification.4 Furthermore, the potential relationship between the different types of calcification is poorly understood. The role of vascular calcification in atherosclerosis remains under discussion.1,4 Even though it is accepted as a marker for the atherosclerotic plaque burden in coronaries and has been associated with an increased risk for cardiovascular morbidity and mortality,1–5 its activity in the disease process has been questioned. On the one hand, it may limit plaque growth and represent a response to injury similar …

Regulation of calcification by bisphosphonates

Etidronate, an analogue of first generation of bisphosphonates, has inhibitory actions on mineralization and hydroxyapatite crystal formation, which is therefore used as a drug to treat ectopic ossification in cases with total hip arthroplasty and spinal cord injury. The effect of bisphosphonates to prevent calcification could be associated with the physical property to attach tightly to hydroxyapatite crystals in bone matrix. A recent investigation suggested that bisphosphonates might prevent arterial calcification through a mechanism associated with the enhanced production of parathyroid hormone-related peptide from vascular smooth muscle cells. A derivative of bisphosphonates to inhibit vascular calcification in atherosclerosis is expected and should be developed in the near future.

Cholesterol in Vascular and Valvular Calcification

In the early 1990s, the late Jeff Hoeg had the foresight to ask whether vascular calcification relates to the duration and severity of exposure to cholesterol. In a group of homozygous hyperlipidemic patients for whom detailed records of cholesterol levels were available over a long period of time, he and his colleagues found that coronary calcification scores by ultrafast computed tomographic scanning correlated significantly with the cholesterol year product.1 Studies that lack long-term cholesterol history may miss a correlation between lipids and calcification, because current treatments so dramatically change cholesterol levels as to make them unrepresentative of long-term exposure. See p 1927 In the present issue of Circulation , Pohle et al2 successfully overcame these difficulties by studying the rate of change in calcification as a function of change in lipid levels, excluding patients in whom treatment was altered during the study period. These investigators found that both coronary calcification and aortic valve calcification progress more rapidly in subjects with levels of LDL >130 mg/dL. This finding dovetails with those of Callister et al,3 who discovered that patients who successfully lowered their cholesterol levels with lipid-lowering agents significantly reduced the progression of coronary calcification. In a large population of young subjects, the risk factor that correlated most significantly with coronary calcification was LDL cholesterol.4 This relation was stronger than the correlation with age, smoking, fasting insulin, blood pressure, or male sex.4 In another study of asymptomatic outpatients, LDL cholesterol was the only risk factor correlating with progression of coronary calcification by electron-beam computed tomography over an 18-month period.5 The correlation of LDL cholesterol exposure to valvular and vascular calcification raises the possibility of a mechanistic role, which is also supported by in vitro studies. Two groups …

Regulation of vascular calcification in atherosclerosis.

Over a century ago it was recognized that the vessel wall is a predominant site for ectopic calcification which is a hallmark of clinically significant atherosclerotic lesions. Old observational studies, which characterized vascular calcification as osteogenesis, and recent identification of common molecular mechanisms in bone and vascular calcification have led to the new recognition that atherosclerotic calcification is an actively regulated process similar to osteogenesis and distinct from a metastatic passive mineralization. Since the atherosclerotic lesion is composed of a multitude of cells and inflammatory mediators, elucidation of the role of these components in induction and acceleration of calcification is of fundamental importance in better understanding its pathogenesis and identifying possible interventional targets. This article will focus on four important mediators of vascular calcification: 1) calcifying vascular cells, 2) oxidized lipids, 3) cytokines, and 4) leptin.

Effects of lectins on calcification by vesicles isolated from aortas of cholesterol-fed rabbits

Abstract Advanced vascular calcification in atherosclerosis weakens arterial walls, thereby imposing a serious rupturing effect. However, the mechanism of dystrophic calcification remains unknown. Although accumulating morphological and biochemical evidence reveals a role for calcifiable vesicles in plaque calcification, the mechanism of vesicle-mediated calcification has not been fully explored. To study whether vesicles’ membrane components, such as carbohydrates, may have a role in vesicle-mediated calcification, the effect of sugar-binding lectins on calcification was investigated. Atherosclerosis was developed by feeding rabbits with a diet supplemented with 0.5% cholesterol and 2% peanut oil for 4 months. Calcifiable vesicles were then isolated from thoracic aortas by collagenase digestion. The histological examination of aortas with hematoxylin counter-staining indicated abnormal formation of large plaques enriched with macrophage-derived foam cells. Fourier transform spectroscopy revealed mild calcification in aortas indicating that advanced stages of heavy calcification have yet to be reached. However, vesicles isolated from the aortas were capable of calcification in the presence of physiological levels of Ca 2+ , Pi, and ATP. Thus, at this stage of atherosclerosis, aortas may start to produce calcifiable vesicles, but at a level insufficient for substantial formation of mineral in aortas. The assessments by FT-IR analysis and Alizarin red staining indicated that concanavalin A (Con A) substantially increased mineral formation by isolated vesicles. Con A also exerted a marked stimulatory effect on 45 Ca and 32 Pi deposition in a dose-dependent fashion with a half-maximal effect at 6–10 μg/ml. Either α-methylmannoside or α-methylglucoside, but not mannitol, at 10 mM abolished the stimulation. Con A stimulation was abolished after Con A was removed from calcifying media, suggesting that covalent binding may not be involved in the effect. Galactosides appear to also be implicated in 45 Ca and 32 Pi deposition since Abrus precartorius agglutinin, which specifically binds galactosides, enhanced the deposition. Neither wheat-germ agglutinin that binds N -acetylglucoside nor N -acetylgalactoside-specific Helix pomatia agglutinin was effective, suggesting that the acetylated forms of carbohydrate moieties are either absent in vesicles or may not be involved in calcification. None of these lectins exerted an effect on ATPase. Thus, the effects of lectins appeared to be mediated through interactions with carbohydrate moieties of calcifiable vesicles. Whether stimulation of vesicle-calcification by lectins is of pathological significance in atherosclerotic calcification requires further investigation.

Effects of lectins on calcification by vesicles isolated from aortas of cholesterol-fed rabbits

Advanced vascular calcification in atherosclerosis weakens arterial walls, thereby imposing a serious rupturing effect. However, the mechanism of dystrophic calcification remains unknown. Although accumulating morphological and biochemical evidence reveals a role for calcifiable vesicles in plaque calcification, the mechanism of vesicle-mediated calcification has not been fully explored. To study whether vesicles’ membrane components, such as carbohydrates, may have a role in vesicle-mediated calcification, the effect of sugar-binding lectins on calcification was investigated. Atherosclerosis was developed by feeding rabbits with a diet supplemented with 0.5% cholesterol and 2% peanut oil for 4 months. Calcifiable vesicles were then isolated from thoracic aortas by collagenase digestion. The histological examination of aortas with hematoxylin counter-staining indicated abnormal formation of large plaques enriched with macrophage-derived foam cells. Fourier transform spectroscopy revealed mild calcification in aortas indicating that advanced stages of heavy calcification have yet to be reached. However, vesicles isolated from the aortas were capable of calcification in the presence of physiological levels of Ca 2+, Pi, and ATP. Thus, at this stage of atherosclerosis, aortas may start to produce calcifiable vesicles, but at a level insufficient for substantial formation of mineral in aortas. The assessments by FT-IR analysis and Alizarin red staining indicated that concanavalin A (Con A) substantially increased mineral formation by isolated vesicles. Con A also exerted a marked stimulatory effect on 45Ca and 32Pi deposition in a dose-dependent fashion with a half-maximal effect at 6–10 μg/ml. Either α-methylmannoside or α-methylglucoside, but not mannitol, at 10 mM abolished the stimulation. Con A stimulation was abolished after Con A was removed from calcifying media, suggesting that covalent binding may not be involved in the effect. Galactosides appear to also be implicated in 45Ca and 32Pi deposition since Abrus precartorius agglutinin, which specifically binds galactosides, enhanced the deposition. Neither wheat-germ agglutinin that binds N-acetylglucoside nor N-acetylgalactoside-specific Helix pomatia agglutinin was effective, suggesting that the acetylated forms of carbohydrate moieties are either absent in vesicles or may not be involved in calcification. None of these lectins exerted an effect on ATPase. Thus, the effects of lectins appeared to be mediated through interactions with carbohydrate moieties of calcifiable vesicles. Whether stimulation of vesicle-calcification by lectins is of pathological significance in atherosclerotic calcification requires further investigation.