Cell differentiation in vascular calcification

Summary Ectopic tissue formation is commonly found in calcified atherosclerotic plaques. This suggests that cell differentiation plays an important role in vascular calcification, even though the origin of the cells involved is unclear. Calcifying vascular cells (CVCs), derived from bovine aortic media, have been used as an in vitro model for vascular calcification. CVCs have many characteristics in common with bone cells, but there are also differences suggesting mechanisms that may be applicable to the problem of osteoporosis in the setting of vascular calcification. Matrix GLA protein (MGP) deficient mice develop severe vascular calcification and die prematurely from heart failure and/or aortic rupture. The molecular mechanism of MGP is unknown. It has been hypothesized that MGP acts as a calcification inhibitor by binding calcium, preventing mineral deposition in extracelular fluids near the saturation point for calcium and phosphate. Alternatively, MGP expression may be an attempt to regulate cell differentiation in the vascular wall, possibly by acting as an inhibitor to a factor able to induce cartilage and bone such as bone morphogenetic proteins (BMPs).     

Endothelial cells modulate osteogenesis in calcifying vascular cells

The potential role of vascular endothelium in atherosclerotic calcification is unknown. Endothelial cells (EC) express bone morphogenetic proteins (BMP), and EC-conditioned medium is osteoinductive in marrow stromal cells. To test whether EC are osteoinductive in vascular cells, we used calcifying vascular cells (CVC) that form nodules and mineralize in vitro. We established a coculture model with EC grown opposite CVC on membranes coated with collagen I or collagen IV, both of which are expressed in atherosclerotic lesions. On collagen I, EC did not alter CVC nodule formation, calcification or expression of the osteogenic marker Cbfa1, the chondrogenic marker collagen IX or smooth muscle cell alpha-actin. However, on collagen IV, EC abolished nodule formation and calcification, and expression of cell markers decreased, suggesting dedifferentiation. Matrix GLA protein (MGP), also expressed in atherosclerotic lesions, was added to CVC in coculture. Unexpectedly, MGP enhanced Cbfa1 expression in CVC on both collagen I and IV. The enhancement was most apparent on collagen IV, where calcification also increased. However, MGP did not restore nodule formation on collagen IV, suggesting that nodule formation and cell differentiation are separate processes. The effect of EC on CVC calcification was suppressed by noggin, an inhibitor of BMP activity, and in part mimicked by replacement of EC by BMP-2. Our results support a role for endothelium in vascular calcification, modulated by collagens and MGP.     

Proline and gamma-carboxylated glutamate residues in matrix Gla protein are critical for binding of bone morphogenetic protein-4

Arterial calcification is ubiquitous in vascular disease and is, in part, prevented by matrix Gla protein (MGP). MGP binds calcium ions through gamma-carboxylated glutamates (Gla residues) and inhibits bone morphogenetic protein (BMP)-2/-4. We hypothesized that a conserved proline (Pro)64 is essential for BMP inhibition. We further hypothesized that calcium binding by the Gla residues is a prerequisite for BMP inhibition. Site-directed mutagenesis was used to modify Pro64 and the Gla residues, and the effect on BMP-4 activity, and binding of BMP-4 and calcium was tested using luciferase reporter gene assays, coimmunoprecipitation, crosslinking, and calcium quantification. The results showed that Pro64 was critical for binding and inhibition of BMP-4 but not for calcium binding. The Gla residues were also required for BMP-4 binding but flexibility existed. As long as 1 Gla residue remained on each side of Pro64, the ability to bind and inhibit BMP-4 was preserved. Chelation of calcium ions by EDTA or warfarin treatment of cells led to loss of ability of MGP to bind BMP-4. Our results also showed that phenylalanine could replace Pro64 without loss of function and that zebrafish MGP, which lacks upstream Gla residues, did not function as a BMP inhibitor. The effect of MGP mutagenesis on vascular calcification was determined in calcifying vascular cells. Only MGP proteins with preserved ability to bind and inhibit BMP-4 prevented osteogenic differentiation and calcification. Together, our results suggest that BMP and calcium binding in MGP are independent but functionally intertwined processes and that the BMP binding is essential for prevention of vascular calcification.     

Receptor tyrosine kinase Axl modulates the osteogenic differentiation of pericytes

Vascular pericytes undergo osteogenic differentiation in vivo and in vitro and may, therefore, be involved in diseases involving ectopic calcification and osteogenesis. The purpose of this study was to identify factors that inhibit the entry of pericytes into this differentiation pathway. RNA was prepared from pericytes at confluence and after their osteogenic differentiation (mineralized nodules). Subtractive hybridization was conducted on polyA PCR-amplified RNA to isolate genes expressed by confluent pericytes that were downregulated in the mineralized nodules. The subtraction product was used to screen a pericyte cDNA library and one of the positive genes identified was Axl, the receptor tyrosine kinase. Northern and Western blotting confirmed that Axl was expressed by confluent cells and was downregulated in mineralized nodules. Western blot analysis demonstrated that confluent pericytes also secrete the Axl ligand, Gas6. Immunoprecipitation of confluent cell lysates with an anti-phosphotyrosine antibody followed by Western blotting using an anti-Axl antibody, demonstrated that Axl was active in confluent pericytes and that its activity could not be further enhanced by incubating the cells with recombinant Gas6. The addition of recombinant Axl-extracellular domain (ECD) to pericyte cultures inhibited the phosphorylation of Axl by endogenous Gas6 and enhanced the rate of nodule mineralization. These effects were inhibited by coincubation of pericytes with Axl-ECD and recombinant Gas6. Together these results demonstrate that activation of Axl inhibits the osteogenic differentiation of vascular pericytes.     

N-3 fatty acids inhibit vascular calcification via the p38-mitogen-activated protein kinase and peroxisome proliferator-activated receptor-gamma pathways

Fish oil supplementation is associated with lower risk of coronary artery disease in humans, and it has been shown to reduce ectopic calcification in an animal model. However, whether N-3 fatty acids, active ingredients of fish oil, have direct effects on calcification of vascular cells is not clear. In this report, we investigated the effects of eicosapentaenoic acid and docosahexaenoic acid (DHA) on osteoblastic differentiation and mineralization of calcifying vascular cells (CVCs), a subpopulation of bovine aortic medial cells that undergo osteoblastic differentiation and form calcified matrix in vitro. Results showed that N-3 fatty acids inhibited alkaline phosphatase (ALP) activity and mineralization of vascular cells, suggesting that they directly affect osteoblastic differentiation in vascular cells. By Western blot analysis, DHA activated p38-mitogen-activated protein kinase (MAPK) but not extracellular-regulated kinase (ERK) or Akt. An inhibitor of p38-MAPK partially reversed the inhibitory effects of DHA on osteoblastic differentiation and mineralization. Transient transfection experiments showed that DHA also activated peroxisome proliferator-activated receptor-gamma (PPAR-gamma). Both p38-MAPK activator and PPAR-gamma agonists reproduced the inhibitory effects of DHA on CVC mineralization. Pretreatment with DHA also inhibited interleukin-6-induced ALP activity and mineralization. Together, these results suggest that N-3 fatty acids directly inhibit vascular calcification, and that the inhibitory effects are mediated by the p38-MAPK and PPAR-gamma pathways.     

Current concepts of vascular calcification

Vascular calcification, such as coronary and aortic calcification, is a significant feature of vascular pathology. Two distinct forms of vascular calcification are well recognized. One is medial calcification, which occurs between the cell layers of smooth muscle cells, and is related to aging, diabetes and chronic renal failure. The other is atherosclerotic calcification, which occurs in the intima during the development of atheromatous disease. It has been shown that statins inhibit the progression of calcification in the aortic valve and the coronary artery. We have found that statins inhibit calcification of human aortic smooth muscle cells, which is induced by incubating the cells in high-phosphate medium. We also found that this is mediated by inhibiting cellular apoptosis, an essential mechanism for calcification, not by inhibiting inorganic phosphate (Pi) uptake by sodium-dependent phosphate cotransporter (NPC). Besides apoptosis and Pi uptake, such proteins as osteoprotegerin (OPG), matrix Gla protein (MGP), Klotho, fetuin-A, and apoE have been shown to negatively affect vascular calcification. Many previous reports suggest that vascular calcification appears to be regulated by promoting factors, such as Pi, apoptosis, modified LDL, advanced glycation end products, oxidative stress, vitaminD3, glucocorticoid, cbfa-1, osteopontin, and inhibitory factors, such as OPG, MGP, Klotho, fetuin-A, PTH/PTHrP, pyrophosphate, statins, and bisphosphonates. The precise mechanism of vascular calcification is of interest.     

The regulation of valvular and vascular sclerosis by osteogenic morphogens

Vascular calcification increasingly afflicts our aging, dysmetabolic population. Once considered only a passive process of dead and dying cells, vascular calcification has now emerged as a highly regulated form of biomineralization organized by collagenous and elastin extracellular matrices. During skeletal bone formation, paracrine epithelial-mesenchymal and endothelial-mesenchymal interactions control osteochondrocytic differentiation of multipotent mesenchymal progenitor cells. These paracrine osteogenic signals, mediated by potent morphogens of the bone morphogenetic protein and wingless-type MMTV integration site family member (Wnt) superfamilies, are also active in the programming of arterial osteoprogenitor cells during vascular and valve calcification. Inflammatory cytokines, reactive oxygen species, and oxylipids-increased in the clinical settings of atherosclerosis, diabetes, and uremia that promote arteriosclerotic calcification-elicit the ectopic vascular activation of osteogenic morphogens. Specific extracellular and intracellular inhibitors of bone morphogenetic protein-Wnt signaling have been identified as contributing to the regulation of osteogenic mineralization during development and disease. These inhibitory pathways and their regulators afford the development of novel therapeutic strategies to prevent and treat valve and vascular sclerosis.     

Osteopontin: a multifunctional molecule regulating chronic inflammation and vascular disease

Osteopontin (OPN) is a multifunctional molecule highly expressed in chronic inflammatory and autoimmune diseases, and it is specifically localized in and around inflammatory cells. OPN is a secreted adhesive molecule, and it is thought to aid in the recruitment of monocytes-macrophages and to regulate cytokine production in macrophages, dendritic cells, and T-cells. OPN has been classified as T-helper 1 cytokine and thus believed to exacerbate inflammation in several chronic inflammatory diseases, including atherosclerosis. Besides proinflammatory functions, physiologically OPN is a potent inhibitor of mineralization, it prevents ectopic calcium deposits and is a potent inducible inhibitor of vascular calcification. Clinically, OPN plasma levels have been found associated with various inflammatory diseases, including cardiovascular burden. It is thus imperative to dissect the OPN proinflammatory and anticalcific functions. OPN recruitment functions of inflammatory cells are thought to be mediated through its adhesive domains, especially the arginine-glycine-aspartate (RGD) sequence that interacts with several integrin heterodimers. However, the integrin receptors and intracellular pathways mediating OPN effects on immune cells are not well established. Furthermore, several studies show that OPN is cleaved by at least 2 classes of proteases: thrombin and matrix-metalloproteases (MMPs). Most importantly, at least in vitro, fragments generated by cleavage not only maintain OPN adhesive functions but also expose new active domains that may impart new activities. The role for OPN proteolytic fragments in vivo is almost completely unexplored. We believe that further knowledge of the effects of OPN fragments on cell responses might help in designing therapeutics targeting inflammatory and cardiovascular diseases.     

Mechanism of vascular calcification

Vascular calcification, especially in coronary artery not only serves as a surrogate marker for atherosclerosis, but also predicts a higher risk of myocardial infarction and death. Its pathogenesis involves an active mineralization process by chondrogenic and osteogenic cells as well as a passive precipitation of minerals. Since atherosclerosis is a chronic vascular inflammation, plaque calcification is conceptualized as a chondrogenic and/or osteogenic process associated with chronic vascular inflammation.     

Role of vitamin K and vitamin K-dependent proteins in vascular calcification

Summary Objectives. To provide a rational basis for recommended daily allowances (RDA) of dietary phylloquinone (vitamin K₁) and menaquinone (vitamin K₂) intake that adequately supply extrahepatic (notably vascular) tissue requirements. Background. Vitamin K has a key function in the synthesis of at least two proteins involved in calcium and bone metabolism, namely osteocalcin and matrix Gla-protein (MGP). MGP was shown to be a strong inhibitor of vascular calcification. Present RDA values for vitamin K are based on the hepatic phylloquinone requirement for coagulation factor synthesis. Accumulating data suggest that extrahepatic tissues such as bone and vessel wall require higher dietary intakes and have a preference for menaquinone rather than for phyloquinone. Methods. Tissue-specific vitamin K consumption under controlled intake was determined in warfarin-treated rats using the vitamin K-quinone/epoxide ratio as a measure for vitamin K consumption. Immunohistochemical analysis of human vascular material was performed using a monoclonal antibody against MGP. The same antibody was used for quantification of MGP levels in serum. Results. At least some extrahepatic tissues including the arterial vessel wall have a high preference for accumulating and using meaquinone rather than phylloquinone. Both intima and media sclerosis are associated with high tissue concentrations of MGP, with the most prominent accumulation at the interface between vascular tissue and calcified material. This was consistent with increased concentrations of circulating MGP in subjects with atherosclerosis and diabetes mellitus. Conclusions. This is the first report demonstrating the association between MGP and vascular calcification. The hypothesis is put forward that undercarboxylation of MGP is a risk factor for vascular calcification and that the present RDA values are too low to ensure full carboxylation of MGP.     

Thyroid hormone targets matrix Gla protein gene associated with vascular smooth muscle calcification

Thyroid hormones have marked cardiovascular effects in vivo. However, their direct effects on vascular smooth muscle cells have been unclear. Because thyroid hormones play critical roles in bone remodeling, we hypothesized that they are also associated with vascular smooth muscle calcification, one of the pathological features of vascular sclerosis. To test this hypothesis, we examined the effects of 3',3,5-triiodo-L-thyronine (T3) on the expression of calcification-associated genes in rat aortic smooth muscle cells (RAOSMCs). Quantitative RT-PCRs revealed that a physiological concentration of T3 (15 pmol/L free T3) increased mRNA level of matrix Gla protein (MGP), which acts as a potent inhibitor of vascular calcification in vivo, by 3-fold in RAOSMCs, as well as in cultured human coronary artery smooth muscle cells. In RAOSMCs transiently transfected with a luciferase reporter gene driven by the MGP promoter, T3 significantly stimulated luciferase activity. In addition, RNA interference against thyroid hormone receptor-alpha gene diminished the effect of T3 on MGP expression. Aortic smooth muscle tissues from methimazole-induced hypothyroid rats (400 mg/L drinking water; 4 weeks) also showed a 68% decrease in the MGP mRNA level, as well as a 33% increase in calcium content compared with that from the control euthyroid animals, whereas hyperthyroidism (0.2 mg T3/kg IP; 10 days) upregulated MGP mRNA by 4.5-fold and reduced calcium content by 11%. Our findings suggest that a physiological concentration of thyroid hormone directly facilitates MGP gene expression in smooth muscle cells via thyroid hormone nuclear receptors, leading to prevention of vascular calcification in vivo.     

Vascular calcification: expression patterns of the osteoblast-specific gene core binding factor alpha-1 and the protective factor matrix gla protein in human atherogenesis.

OBJECTIVE: Increasing evidence suggests that vascular calcification is a regulated process. We studied the vascular expression pattern of a key factor in mineralization and a counteracting, protective factor. Based on the phenotype of null mice, Core binding factor alpha-1 (Cbfa-1) plays a pivotal role in bone formation, whereas Matrix Gla Protein (MGP) is a potent inhibitor of vascular calcification. METHODS: We investigated the expression of MGP and Cbfa-1 in cultured, human monocytic cells, endothelial cells and smooth muscle cells (SMC), as well as in normal and atherosclerotic vessel specimens. RESULTS: In cultured cells MGP is expressed in endothelial cells and SMC, whereas Cbfa-1 mRNA is predominantly present in macrophages and to a lesser extent in SMC. In the normal vessel wall MGP expression is high at the luminal side and declines toward the center of the media, whereas Cbfa-1 is absent. Moderate, diffuse calcification of the aorta media was observed only in those regions where MGP is low or absent. In atherosclerotic lesions MGP is expressed in endothelial cells and SMC that form fibrous caps, but is never present in macrophages. Cbfa-1 is synthesized in regions without MGP, it is associated with calcified areas and Cbfa-1 may be considered a marker for osteoprogenitor-like cells in the vessel wall. CONCLUSIONS: Our observations on MGP expression confirm and extend published data and are consistent with a protective function of MGP. Cbfa-1 expression is absent in normal medial SMC and co-localizes with neointimal macrophages and focal calcifications.     

Pathophysiology of vascular calcification in chronic kidney disease

Patients with chronic kidney disease (CKD) on dialysis have 2- to 5-fold more coronary artery calcification than age-matched individuals with angiographically proven coronary artery disease. In addition to increased traditional risk factors, CKD patients also have a number of nontraditional cardiovascular risk factors that may play a prominent role in the pathogenesis of arterial calcification, including duration of dialysis and disorders of mineral metabolism. In histological specimens from the inferior epigastric artery of dialysis patients, we have found expression of the osteoblast differentiation factor core binding factor alpha-1 (Cbfa1) and several bone-associated proteins (osteopontin, bone sialoprotein, alkaline phosphatase, type I collagen) in both the intima and medial layers when calcification was present. In cultured vascular smooth muscle cells, the addition of pooled serum from dialysis patients (versus normal healthy controls) accelerated mineralization and increased expression of Cbfa1, osteopontin, and alkaline phosphatase to a similar magnitude as does beta-glycerophosphate alone. However, a lack of inhibitors of calcification may also be important. Dialysis patients with low levels of serum fetuin-A, a circulating inhibitor of mineralization, have increased coronary artery calcification and fetuin-A can inhibit mineralization of vascular smooth muscle cells in vitro. These data support that elevated levels of phosphorus and/or other potential uremic toxins may play an important role by transforming vascular smooth muscle cells into osteoblast-like cells, which can produce a matrix of bone collagen and noncollagenous proteins. This nidus can then mineralize if the balance of pro-mineralizing factors outweighs inhibitory factors.     

Regulation of vascular calcification by osteoclast regulatory factors RANKL and osteoprotegerin

Vascular calcification often occurs with advancing age, atherosclerosis, various metabolic disorders such as diabetes mellitus and end-stage renal disease, or in rare genetic diseases, leading to serious clinical consequences. Such mineralization can occur at various sites (cardiac valves, arterial intima or media, capillaries), involve localized or diffuse widespread calcification, and result from numerous causes that provoke active inflammatory and osteogenic processes or disordered mineral homeostasis. Although valuable research has defined many key factors and cell types involved, surprising new insights continue to arise that deepen our understanding and suggest novel research directions or strategies for clinical intervention in calcific vasculopathies. One emerging area in vascular biology involves the RANKL/RANK/OPG system, molecules of the tumor necrosis factor-related family recently discovered to be critical regulators of immune and skeletal biology. Evidence is accumulating that such signals may be expressed, regulated, and function in vascular physiology and pathology in unique ways to promote endothelial cell survival, angiogenesis, monocyte or endothelial cell recruitment, and smooth muscle cell osteogenesis and calcification. Concerted research efforts are greatly needed to understand these potential roles, clarify whether RANKL (receptor activator of nuclear factor kappaB ligand) promotes and osteoprotegerin (OPG) protects against vascular calcification, define how OPG genetic polymorphisms relate to cardiovascular disease, and learn whether elevated serum OPG levels reflect endothelial dysfunction in patients. Overall, the RANKL/RANK/OPG system may mediate important and complex links between the vascular, skeletal, and immune systems. Thus, these molecules may play a central role in regulating the development of vascular calcification coincident with declines in skeletal mineralization with age, osteoporosis, or disease.     

Vitamin K and vascular calcification

Until recently, vitamin K has been exclusively related to blood coagulation. During the last decade, a second function for vitamin K-dependent proteins has become apparent : the regulation of tissue calcification. One of them is the function of Matrix Gla Protein (MGP) : potent inhibitors of vascular calcification. The function of MGP became clear from transgenic mice (MGP-deficient mice). Further research on MGP will resolve the complicated mechanism of atherosclerosis, especially of the arterial calcification. The recommended daily allowance for vitamin K to prevent vascular calcification should be evaluated.     

血管钙化参与细胞相关研究的新进展

血管钙化是一个受细胞和基因主动调节的过程,涉及血管钙化促进因子和抑制因子的平衡失调。血管钙化常见于动脉粥样硬化晚期,与糖尿病、慢性肾病及衰老等疾病密切相关。血管细胞的成骨样分化是血管钙化的关键环节,但参与血管钙化调节的细胞来源尚不十分明确。目前认为调节血管钙化的细胞来源可能包括:血管平滑肌细胞、内皮细胞、周细胞、巨噬细胞及祖细胞等。本文现将相关进展作一简要综述。