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.

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.

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.

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.

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.

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.

Atherogenic diet and minimally oxidized low density lipoprotein inhibit osteogenic and promote adipogenic differentiation of marrow stromal cells.

In osteoporosis, the bone marrow stroma osteogenic cell population declines and adipocyte numbers increase. We recently showed that oxidized lipids inhibit differentiation of preosteoblasts. In this report, we assess the effect of minimally oxidized low density lipoprotein (MM-LDL) on osteoblastic differentiation of murine marrow stromal cells, M2-10B4. MM-LDL, but not native LDL, inhibited stromal cell osteoblastic differentiation as demonstrated by inhibition of alkaline phosphatase activity, collagen I processing, and mineralization, through a mitogen-activated protein kinase-dependent pathway. In addition, marrow stromal cells from C57BL/6 mice fed a high fat, atherogenic diet failed to undergo osteogenic differentiation in vitro. The ability of MM-LDL to regulate adipogenesis was also assessed. Treatment of M2-10B4 as well as 3T3-L1 preadipocytes with MM-LDL, but not native LDL, promoted adipogenic differentiation in the presence of peroxisome proliferator-activated receptor (PPAR) gamma agonist thiazolidinediones, BRL49653 and ciglitizone. Based on promoter-reporter construct experiments, MM-LDL may be acting in part through activating PPARalpha. These observations suggest that LDL oxidation products promote osteoporotic loss of bone by directing progenitor marrow stromal cells to undergo adipogenic instead of osteogenic differentiation. These data lend support to the "lipid hypothesis of osteoporosis."

cAMP stimulates osteoblast-like differentiation of calcifying vascular cells. Potential signaling pathway for vascular calcification.

The role of the cAMP signaling pathway in vascular calcification was investigated using calcifying vascular cells (CVC) derived from primary aortic medial cell cultures. We previously showed that CVC have fibroblastic morphology and express several osteoblastic differentiation markers. After confluency, they aggregate into cellular condensations, which later mature into nodules where mineralization is localized. Here, we investigated the effects of cAMP on CVC differentiation because it plays a role in both osteoblastic differentiation and vascular disease. Dibutyryl-cAMP or forskolin treatment of CVC for 3 days induced osteoblast-like "cuboidal" morphology, inhibited proliferation, and enhanced alkaline phosphatase activity, all early markers of osteoblastic differentiation. Isobutylmethylxanthine and cholera toxin had the same effects. Treatment of CVC with pertussis toxin, however, did not induce the morphological change or increase alkaline phosphatase activity, although it inhibited CVC proliferation to a similar extent. cAMP also increased type I procollagen production and gene expression of matrix gamma-carboxyglutamic acid protein, recently shown to play a role in in vivo vascular calcification. cAMP inhibited the expression of osteopontin but did not affect the expression of osteocalcin and core binding factor. Prolonged cAMP treatment enhanced matrix calcium-mineral incorporation but inhibited the condensations resulting in diffuse mineralization throughout the monolayer of cells. Treatment of CVC with a protein kinase A-specific inhibitor, KT5720, inhibited alkaline phosphatase activity and mineralization during spontaneous CVC differentiation. These results suggest that the cAMP pathway promotes in vitro vascular calcification by enhancing osteoblast-like differentiation of CVC.

Lipid oxidation products have opposite effects on calcifying vascular cell and bone cell differentiation. A possible explanation for the paradox of arterial calcification in osteoporotic patients.

Atherosclerotic calcification and osteoporosis often coexist in patients, yielding formation of bone mineral in vascular walls and its simultaneous loss from bone. To assess the potential role of lipoproteins in both processes, we examined the effects of minimally oxidized low-density lipoprotein (MM-LDL) and several other lipid oxidation products on calcifying vascular cells (CVCs) and bone-derived preosteoblasts MC3T3-E1. In CVCs, MM-LDL but not native LDL inhibited proliferation, caused a dose-dependent increase in alkaline phosphatase activity, which is a marker of osteoblastic differentiation, and induced the formation of extensive areas of calcification. Similar to MM-LDL, oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (ox-PAPC) and the isoprostane 8-iso prostaglandin E2 but not PAPC or isoprostane 8-iso prostaglandin F2 alpha induced alkaline phosphatase activity and differentiation of CVCs. In contrast, MM-LDL and the above oxidized lipids inhibited differentiation of the MC3T3-E1 bone cells, as evidenced by their stimulatory effect on proliferation and their inhibitory effect on the induction of alkaline phosphatase and calcium uptake. These results suggest that specific oxidized lipids may be the common factors underlying the pathogenesis of both atherosclerotic calcification and osteoporosis.

PCR mutagenesis identifies a polymerase-binding sequence of sigma 54 that includes a sigma 70 homology region.

Sigma 54 is a minor bacterial sigma factor that is not a member of the sigma 70 family of proteins but binds the same core RNA polymerase. Previously, we identified a region of sigma 54 that is important for binding core polymerase. In this work, PCR mutagenesis was used to identify specific amino acids important for this binding. The results show that important residues are clustered most closely in a short sequence that was previously speculated to be potentially homologous to a sequence in sigma 70. The mutagenesis also identifies important residues in the flanking hydrophobic-acidic region of sigma 54, which is absent in sigma 70. Overall, the data indicate that sigma 54 binds core polymerase through a sequence homologous to that of sigma 70 but in addition uses unique motifs to modify this interaction.

Abortive cycling and the release of polymerase for elongation at the sigma 54-dependent glnAp2 promoter.

Transcription initiation at the sigma 54-dependent glnAp2 promoter was studied to follow the state of polymerase as RNA synthesis begins. Sigma 54 polymerase begins transcription in abortive cycling mode, i.e. after the first bond is made, approximately 75% of the time the short RNA is aborted and synthesis must be restarted. Polymerase is capable of abortive initiation until it reaches a position beyond +3 and before +7, at which stage polymerase is released from its promoter contacts and an elongation complex is formed. INitial elongation is accompanied by two transcription bubbles, one moving with the polymerase and the other remaining at the transcription start site. The sigma 54-associated polymerase shows an earlier and more efficient transition out of abortive initiation mode than prior studies of sigma 70-associated polymerase.

A novel bacterial transcription cycle involving sigma 54.

sigma 54 is the promoter recognition subunit of the form of bacterial RNA polymerase that transcribes from promoters with enhancer elements. DNase footprinting experiments show that sigma 54 is attached selectively to the template strand, which must be single-stranded for transcription initiation. sigma 54 remains bound at the promoter after core polymerase begins elongation, in contrast to the well-established sigma 70-holoenzyme transcription cycle. Permanganate footprinting experiments show that the bound sigma 54 and the elongating core RNA polymerase downstream of it are each associated with a single-strand DNA region. Template commitment assays show that the promoter-bound sigma 54 must be reconfigured before reinitiation of transcription can occur. This unexpected pathway raises interesting possibilities for transcriptional regulation, especially with regard to control at the level of reinitiation.

RNA polymerase binding using a strongly acidic hydrophobic-repeat region of sigma 54.

sigma 54 is a rare bacterial protein that substitutes for sigma 70 in the case of Escherichia coli genes transcribed by certain activators with enhancer protein-like properties. It contains a strongly acidic region of previously unknown function. Gel mobility-shift assays using sigma 54 deletion mutants show that this region is essential for sigma 54 to bind the core RNA polymerase and recruit it to the promoter. Multiple-point mutational analysis shows that the acidic amino acids and overlapping periodic hydrophobic amino acids are necessary for this binding. Related sequences are not found within the core binding determinant of sigma 70, which binds the same core RNA polymerase. This comparison suggests that the core RNA polymerase interacts differently with the two sigma factors, likely contributing to the critical differences in transcription mechanism in the two cases.

The domain structure of sigma 54 as determined by analysis of a set of deletion mutants.

Escherichia coli sigma 54 was analyzed by making a series of 16 internal deletions within its gene and analyzing the properties of the mutant proteins. All of the mutant proteins except one were strongly defective in a growth test that relied on sigma 54 function. Additional assays were applied to determine the causes of these defects. The assays monitored the following properties: the level of protein expression; ability to bind to the -24 promoter element of the glnAP2 promoter in vivo; the ability to bind to the -12 promoter element in vivo; ability to melt the promoter start site in vivo; ability to bind the Rhizobium meliloti nifH promoter in vitro; and the ability to form a sigma 54-core RNA polymerase complex (E sigma 54 holoenzyme) in vitro. The analysis shows a modular structure in that certain regions of the protein predominate in contributing to each of these properties. A large carboxyl region of the protein is essential for promoter binding. A smaller amino-terminal segment is essential for DNA melting. An element essential for the forming the E sigma 54 holoenzyme lies between these two regions. None of these domains resemble those of sigma 70 and this difference is discussed in view of the different transcription mechanisms directed by the two proteins.

Functional roles for the glutamines within the glutamine-rich region of the transcription factor sigma 54.

Single and multiple point mutations were introduced to change the 12 glutamine residues within a 37-amino acid region of sigma 54. Multiple changes are shown to be required in order to interfere significantly with the function of this protein which is associated with enhancer-dependent bacterial transcription. Mutation of the central 4 glutamines leads to the production of less m-RNA, caused by an inability to fully open the promoter start site. DNA binding, however, is normal. Mutation of 4 other adjacent glutamines causes the promoter start site to open more readily than wild type, although this enhanced opening is not accompanied by more mRNA. The enhanced DNA melting is not caused by enhanced promoter binding, as indicated by normal protection of the polymerase-bound promoter against dimethyl sulfate attack. The results suggest that multiple glutamines play a role in transducing the melting signal from the enhancer protein to the polymerase.