Enhanced mineralization potential of vascular cells from SM22α-Rankl (tg) mice.

Vascular calcification, prevalent in diabetes and chronic kidney disease, contributes to morbidity and mortality. To investigate the effect of receptor activator of NF-kB ligand (RANKL) on vascular calcification in vivo, transgenic mice, where RANKL expression was targeted to vascular smooth muscle cells using the SM22α promoter (SM22α-Rankl ( tg )), were created. Sixteen-month-old male SM22α-Rankl ( tg ) mice had higher body weight and higher serum calcium levels but lower lumbar bone mineral density (BMD) compared with age- and gender-matched wild-type (WT) littermates. BMD of long bones, body fat (percent of weight) of the leg, and serum levels of phosphate and RANKL were not significantly different. No significant differences in these parameters were observed in female mice. Histological analysis did not reveal calcium deposits in the aortic roots of SM22α-Rankl ( tg ) mice. To analyze the osteoblastic differentiation and mineralization potentials of vascular cells, aortic smooth muscle cells (SMCs) were isolated and cultured. Results showed that SM22α-Rankl ( tg ) SMCs had higher baseline alkaline phosphatase (ALP) activity but not baseline matrix calcification. When induced by the PKA agonist forskolin, ALP activity was greater in SM22α-Rankl ( tg ) than in WT SMCs. Real-time RT-qPCR revealed higher baseline expression of ALP and ankylosis genes but lower osteoprotegerin gene in SM22α-Rankl ( tg ) SMCs. Matrix mineralization induced by inorganic phosphate or forskolin was greater in SM22α-Rankl ( tg ) than in WT SMCs. Treatment of these cells with the ALP inhibitor levamisole abolished forskolin-induced matrix mineralization but not inorganic phosphate-induced matrix mineralization. These findings suggest that RANKL overexpression in the vasculature may promote mineralization potential.

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.

Pulsatile flow regulates monocyte adhesion to oxidized lipid-induced endothelial cells.

Oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (ox-PAPC), a component of minimally modified low density lipoprotein, induces monocyte adhesion to endothelial cells. It is not known whether the upstroke slopes of pulsatile flow, defined as shear stress slew rates (tau(r)/tauT)), can regulate monocyte binding to ox-PAPC-treated bovine aortic endothelial cells (BAECs). At 60 cycles per minute, ox-PAPC-treated BAECs were exposed to 3 conditions representing known vascular conditions: (1) high shear stress slew rates (tau(r)/tau(T)=293 dyne. cm(-2). s(-1)), with time-averaged shear stress=50 dyne/cm(2); (2) low shear stress slew rate (tau(r)/tau(t)=71 dyne. cm(-2). s(-1)), with identical time-averaged shear stress; and (3) reversing oscillating flow (0+/-2.6 mm Hg). Reverse transcription-polymerase chain reaction and quantification were performed for monocyte chemoattractant protein-1 (MCP-1) mRNA expression. High tau(r)/tau(t) reduced monocyte binding to ox-PAPC-treated BAECs by 64+/-3.2% compared with static conditions, and low tau(r)/tau(t) reduced monocyte binding by 31+/-3.4%, whereas oscillating flow increased monocyte binding by 22+/-1.7% (P<0.005). High partial tau(r)/tau(t) downregulated MCP-1 expression by 33+/-8%, and low partial tau(r)/tau(t) downregulated MCP-1 expression by 15+/-4%, but oscillating flow upregulated MCP-1 by 13+/-5%. These results suggest that shear stress slew rates regulate monocyte binding by modulating the expression of a potent monocyte chemoattractant.

A complex flow pattern of low shear stress and flow reversal promotes monocyte binding to endothelial cells.

Predilection sites for atherosclerosis within the vasculature are characterized by low shear stress and flow reversal. In this study, endothelial cells were exposed to a complex flow pattern that was characterized by particle velocity determination. Bovine aortic endothelial cells exposed to low shear stress and flow reversal demonstrated higher levels of monocyte binding compared to endothelial cells exposed to one-directional flow. In addition, endothelial cells exposed to low shear stress and flow reversal responded to inflammatory stimuli with substantial increases in monocyte binding, similar to that seen in cells exposed to one-directional flow. These findings suggest a mechanism by which areas of low shear stress and flow reversal are predisposed to the development of atherosclerotic lesions.

Recent advances in multifactorial regulation of vascular calcification.

Calcification presents important clinical implications in cardiovascular diseases, especially in coronary arteries. Epidemiological evidence has shown the coexistence of vascular calcification with both atherosclerosis and osteoporosis, and increasing evidence has shown the role of hyperlipidemia and atherogenic phospholipids in vascular calcification. The etiology of vascular calcification is also increasingly recognized as an active process. Vascular calcification initiates with matrix vesicle formation and mineralization following a process similar to that in bone. In addition, many bone regulatory factors have been shown to be present in calcified atherosclerotic lesions. In this review, we focus on the new developments emerging during the past year in regulation of vascular calcification. Regulatory factors include matrix GLA protein, the phosphate cotransporter Pit-1, a calcium-sensing receptor related factor, osteoprotegerin, leptin, bisphosphonates and oxidized lipids. Some of these, including oxidized lipids, osteoprotegerin, and bisphosphonates, appear to regulate mineralization in both bone and vasculature and may account for the co-existence of osteoporosis and atherosclerotic calcification that is independent of age.

Oxidative stress modulates osteoblastic differentiation of vascular and bone cells.

Oxidative stress may regulate cellular function in multiple pathological conditions, including atherosclerosis. One feature of the atherosclerotic plaque is calcium mineral deposition, which appears to result from the differentiation of vascular osteoblastic cells, calcifying vascular cells (CVC). To determine the role of oxidative stress in regulating the activity of CVC, we treated these cells with hydrogen peroxide (H(2)O(2)) or xanthine/xanthine oxidase (XXO) and assessed their effects on intracellular oxidative stress, differentiation, and mineralization. These agents increased intracellular oxidative stress as determined by 2,7 dichlorofluorescein fluorescence, and enhanced osteoblastic differentiation of vascular cells, based on alkaline phosphatase activity and mineralization. In contrast, H(2)O(2) and XXO resulted in inhibition of differentiation markers in bone osteoblastic cells, MC3T3-E1, and marrow stromal cells, M2-10B4, while increasing oxidative stress. In addition, minimally oxidized low-density lipoprotein (MM-LDL), previously shown to enhance vascular cell and inhibit bone cell differentiation, also increased intracellular oxidative stress in the three cell types. These effects of XXO and MM-LDL were counteracted by the antioxidants Trolox and pyrrolidinedithiocarbamate. These results suggest that oxidative stress modulates differentiation of vascular and bone cells oppositely, which may explain the parallel buildup and loss of calcification, seen in vascular calcification and osteoporosis, respectively.

Placental calcification: a metastatic process?

Placental calcification commonly increases with gestational age. The mechanism of apatite mineralization probably involves one of three known mechanisms of tissue calcification: physiological (like bone), dystrophic (ischaemia-related) or metastatic (mineralization in a supersaturated environment). This study was designed to determine the mechanism of calcification by examining (1) the mineral content of placental calcifications in comparison to other physiological and pathological apatites, and (2) the expression of bone morphogenetic proteins (BMPs), which are important in physiological calcification, across gestational age. By energy-dispersive x-ray analysis (EDXA), the Ca/P weight ratio for apatitic mineral from mature calcifications was 2.00+/-0.05 (s.e.), which is similar to that for stones formed in a metastatic, supersaturated environment and lower than that observed in physiological calcification. Biologically active BMP, which was determined by bioassay, was demonstrated in mature and postmature placentae. The BMPs PLAB, PDF and related protein INSL-4 were identified by semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR), but their mRNA expression was independent of gestational age (7-41 weeks of gestation). We conclude that (1) the identified BMPs were not related directly to placental calcification, which argues against physiological calcification, and (2) the chemical composition of the apatitic mineral was suggestive of rapid formation in a supersaturated environment, which is consistent with a metastatic mechanism of calcification.

Leptin enhances the calcification of vascular cells: artery wall as a target of leptin.

Leptin, the product of the ob gene, regulates food intake, energy expenditure, and other physiological functions of the peripheral tissues. Leptin receptors have been identified in the hypothalamus and in extrahypothalamic tissues. Increased circulating leptin levels have been correlated with cardiovascular disease, obesity, aging, infection with bacterial lipopolysaccharide, and high-fat diets. All these conditions have also been correlated with increased vascular calcification, a hallmark of atherosclerotic and age-related vascular disease. In addition, the differentiation of marrow osteoprogenitor cells is regulated by leptin. Thus, we hypothesized that leptin may regulate the calcification of vascular cells. In this report, we tested the effects of leptin on a previously characterized subpopulation of vascular cells that undergo osteoblastic differentiation and calcification in vitro. When treated with leptin, these calcifying vascular cells had a significant 5- to 10-fold increase in alkaline phosphatase activity, a marker of osteogenic differentiation of osteoblastic cells. Prolonged treatment with leptin enhanced the calcification of these cells, further supporting the pro-osteogenic differentiation effects of leptin. Furthermore, the presence of the leptin receptor on calcifying vascular cells was demonstrated using reverse transcriptase polymerase chain reaction, immunocytochemistry, and Western blot analysis. We also identified the presence of leptin receptor in the mouse artery wall, localized to subpopulations of medial and adventitial cells, and the expression of leptin by artery wall cells and atherosclerotic lesions in mice. Taken together, these results suggest that leptin regulates the osteoblastic differentiation and calcification of vascular cells and that the artery wall may be an important peripheral tissue target of leptin action.

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.

Matrix GLA protein modulates differentiation induced by bone morphogenetic protein-2 in C3H10T1/2 cells.

Matrix GLA protein (MGP) is ubiquitously expressed with high accumulation in bone and cartilage, where it was found to associate with bone morphogenetic proteins (BMP) during protein purification. To test whether MGP affects BMP-induced differentiation, three sets of experiments were performed. First, pluripotent C3H10T1/2 cells transfected with human MPG (hMGP) or antisense to hMGP (AS-hMGP) were treated with BMP-2. In cells overexpressing hMGP, osteogenic and chondrogenic differentiation was inhibited indicating decreased BMP-2 activity. Conversely, in cells overexpressing AS-hMGP, BMP-2 activity was enhanced. Second, cells were prepared from homozygous and heterozygous MPG-deficient mice aortas. When treated with BMP-2, these cells underwent chondrogenic and osteogenic differentiation, respectively, whereas controls did not. Third, FLAG-tagged hMGP with the same biological effect as native hMGP inhibited BMP-induced differentiation, when exogenously added to culture media. Together, these results suggest that MGP modulates BMP activity. To test whether hMGP fragments would retain the effect of full-length hMGP, three subdomains were overexpressed in C3H10T1/2 cells. In cells expressing the mid-region, alone (amino acids (aa) 35-54) or in combination with the N terminus (aa 1-54) but not the C terminus (aa 35-84), osteogenic differentiation was enhanced and occurred even without added BMP-2. Thus, two subdomains had the opposite effect of full-length hMGP, possibly due to different expression levels or domain characteristics.

Atherogenic high-fat diet reduces bone mineralization in mice.

The epidemiological correlation between osteoporosis and cardiovascular disease is independent of age, but the basis for this correlation is unknown. We previously found that atherogenic oxidized lipids inhibit osteoblastic differentiation in vitro and ex vivo, suggesting that an atherogenic diet may contribute to both diseases. In this study, effects of an atherogenic high-fat diet versus control chow diet on bone were tested in two strains of mice with genetically different susceptibility to atherosclerosis and lipid oxidation. After 4 months and 7 months on the diets, mineral content and density were measured in excised femurs and lumbar vertebrae using peripheral quantitative computed tomographic (pQCT) scanning. In addition, expression of osteocalcin in marrow isolated from the mice after 4 months on the diets was examined. After 7 months, femoral mineral content in C57BL/6 atherosclerosis-susceptible mice on the high-fat diet was 43% lower (0.73 +/- 0.09 mg vs. 1.28 +/- 0.42 mg; p = 0.008), and mineral density was 15% lower compared with mice on the chow diet. Smaller deficits were observed after 4 months. Vertebral mineral content also was lower in the fat-fed C57BL/6 mice. These changes in the atherosclerosis-resistant, C3H/HeJ mice were smaller and mostly not significant. Osteocalcin expression was reduced in the marrow of high fat-fed C57BL/6 mice. These findings suggest that an atherogenic diet inhibits bone formation by blocking differentiation of osteoblast progenitor cells.

Role of lipids in osteoporosis.

Cardiovascular disease and osteoporosis together account for most of the morbidity and mortality in our aging population despite significant improvements in treatment. Recently, converging lines of evidence suggest that these 2 diseases share an etiologic factor--that hyperlipidemia contributes not only to atherosclerotic plaque formation, but also to osteoporosis, following a similar biologic mechanism involving lipid oxidation. In vitro studies indicate that lipid products of oxidation promote osteoblastic differentiation of vascular cells and inhibit such differentiation in bone cells. Ex vivo, in vivo, and clinical studies further suggest that lipid-lowering agents reduce both atherosclerotic calcification and osteoporosis. Whether lipid-lowering agents reduce osteoporosis directly or indirectly through lipid reduction remains controversial.

Tumor necrosis factor-alpha promotes in vitro calcification of vascular cells via the cAMP pathway.

BACKGROUND: Vascular calcification is an ectopic calcification that commonly occurs in atherosclerosis. Because tumor necrosis factor-alpha (TNF-alpha), a pleiotropic cytokine found in atherosclerotic lesions, is also a regulator of bone formation, we investigated the role of TNF-alpha in in vitro vascular calcification. METHODS AND RESULTS: A cloned subpopulation of bovine aortic smooth muscle cells previously shown capable of osteoblastic differentiation was treated with TNF-alpha, and osteoblastic differentiation and mineralization were assessed. Treatment of vascular cells with TNF-alpha for 3 days induced an osteoblast-like morphology. It also enhanced both activity and mRNA expression of alkaline phosphatase, an early marker of osteoblastic differentiation. Continuous treatment with TNF-alpha for 10 days enhanced matrix mineralization as measured by radiolabeled calcium incorporation in the matrix. Pretreatment of cells with a protein kinase A-specific inhibitor, KT5720, attenuated cell morphology, the alkaline phosphatase activity, and mineralization induced by TNF-alpha. Consistent with this, the intracellular cAMP level was elevated after TNF-alpha treatment. Electrophoretic mobility shift assay demonstrated that TNF-alpha enhanced DNA binding of osteoblast specific factor (Osf2), AP1, and CREB, transcription factors that are important for osteoblastic differentiation. CONCLUSIONS: These results suggest that TNF-alpha enhances in vitro vascular calcification by promoting osteoblastic differentiation of vascular cells through the cAMP pathway.

HOXB7 overexpression promotes differentiation of C3H10T1/2 cells to smooth muscle cells.

The presence of immature smooth muscle cells and ectopic tissues such as fully-formed bone in atherosclerotic lesions, may result from recapitulation of embryonic mechanisms in the artery wall. We hypothesized that expression of homeobox genes is triggered in atherogenesis and that these regulate proliferation and differentiation of multipotential progenitor cells along one or more specific lineages. We identified expression of the homeobox gene HOXB7 in clones of bovine aortic medial cells previously shown to be multipotent. HOXB7 was subsequently detected in human atherosclerotic plaques by RT-PCR and in situ hybridization. Expression was localized to areas adjacent to calcification and scattered in media and neointima, which may be reflective of a role in either osteoblastic or smooth muscle cell differentiation. To differentiate between these possibilities, we overexpressed HOXB7 in C3H10T1/2 cells, a multipotent cell line able to differentiate into vascular smooth muscle cells (SMC), as well as osteogenic and chondrogenic lineages. Results showed that overexpression of HOXB7 increased proliferation 3.5-fold, and induced an SMC-like cell morphology. In addition, expression of the early SMC markers calponin and SM22alpha increased 4-fold and 3-fold respectively by semi-quantitative RT-PCR. Expression of the intermediate SMC marker smooth muscle myosin heavy chain (SM-MHC) did not change. No increase in osteogenic or chondrogenic differentiation was detected, neither in the C3H10T1/2 cells nor in M2 cells, a bone marrow stromal cell line used to confirm this result. These findings suggest that HOXB7 plays a role in expansion of immature cell populations or dedifferentiation of mature cells.

Peroxisome proliferator-activated receptor activators modulate the osteoblastic maturation of MC3T3-E1 preosteoblasts.

The reduced bone mineral density (BMD) observed in osteoporosis results, in part, from reduced activity of bone-forming osteoblasts. We examined the effect of peroxisome proliferator-activated receptor (PPAR) activators on MC3T3-E1 preosteoblast maturation. Activators of PPARalpha, delta and gamma induced alkaline phosphatase activity, matrix calcification and the expression of osteoblast genes as determined by reverse transcriptase-polymerase chain reaction. However, at relatively high concentrations of the specific PPARgamma ligands, ciglitazone and troglitazone, maturation was inhibited. PPARalpha, delta and gamma1 were expressed in MC3T3-E1 cells. PPARgamma1 mRNA and protein levels were induced early during osteoblastic maturation. We speculate that endogenous and pharmacological PPAR activators may affect BMD by modulating osteoblastic maturation.

Regulatory mechanisms in vascular calcification.

Vascular calcification is increasingly recognized as a significant contributor to cardiovascular morbidity and mortality as well as a biologically regulated process potentially subject to prevention and reversal. Both coronary and aortic calcification are common and influence plaque rupture, angioplasty and surgical complications, and compensatory enlargement. Aortic calcification increases aortic rigidity and contributes to cadiac ischemia, left ventricular hypertrophy, heart failure, and stroke. Calcification is also common in aortic valve leaflets further compounding adverse hemodynamic effects. Vascular calcification has often been attributed to "passive" crystallization. However, functional similarities between atherosclerotic lesions and bone contradict this view and indicate that it is no more "passive" than in embryonic bone formation or bone repair. Similarities include presence of all the major components of bone osteoid, bone regulatory factors, and subpopulations of artery wall cells that retain osteoblastic lineage potential. Several animal models for vascular calcification are available. Spontaneous vascular calcification occurs in null mice for matrix GLA protein (MGP), a small matrix protein of unknown function, and osteoprotegerin (OPG), known to modulate osteoclast differentiation. Vascular calcification may also be induced by feeding vitamin D and calcium or warfarin to normal animals, or by fat-feeding mice null for apoE or the LDL-receptor. Overall, regulation of vascular calcification is a growing field with surprising mechanisms and connections to other fields of biology.

Inhibition of osteoblast-specific transcription factor Cbfa1 by the cAMP pathway in osteoblastic cells. Ubiquitin/proteasome-dependent regulation.

The cAMP pathway, a major intracellular pathway mediating parathyroid hormone signal, regulates osteoblastic function. Parathyroid hormone (through activation of protein kinase A) has also been shown to stimulate ubiquitin/proteasome activity in osteoblasts. Since the osteoblast-specific transcription factor Osf2/Cbfa1 is important for differentiation of osteoblastic cells, we examined the roles of the cAMP and ubiquitin/proteasome pathways in regulation of Cbfa1. In the osteoblastic cell line, MC3T3-E1, continuous treatment with cAMP elevating agents inhibited both osteoblastic differentiation based on alkaline phosphatase assay and DNA binding ability of Cbfa1 based on a gel retardation assay. Cbfa1 inhibition was paralleled by an inhibitory effect of forskolin on Cbfa1-regulated genes. Northern and Western blot analyses suggested that the inhibition of Cbfa1 by forskolin was mainly at the protein level. Pretreatment with proteasome inhibitors prior to forskolin treatment reversed the effect of forskolin. Furthermore, addition of proteasome inhibitors to forskolin-pretreated samples resulted in recovery of Cbfa1 protein levels and accumulation of polyubiquitinated forms of Cbfa1, indicating a role for the proteasome pathway in the degradation of Cbfa1. These results suggest that suppression of osteoblastic function by the cAMP pathway is through proteolytic degradation of Cbfa1 involving a ubiquitin/proteasome-dependent mechanism.