Background— In diabetes or atherosclerosis, ectopic bone, fat, cartilage, and marrow often develop in arteries. However the mechanism is unknown. We have previously identified a subpopulation of vascular cells (calcifying vascular cells, CVC), derived by dilutional cloning of bovine aortic medial cells, and showed that they undergo osteoblastic differentiation and mineralization. We now show that CVC have the potential to differentiate along other mesenchymal lineages. Methods and Results— To determine the multilineage potential of CVC, molecular and functional markers of multiple mesenchymal lineages were assessed. Chondrogenic potential of CVC was evidenced by expression of types II and IX collagen and cytochemical staining for Alcian blue. Leiomyogenic potential of CVC was evidenced by the expression of smooth muscle-α actin, calponin, caldesmon, and myosin heavy chain. Stromogenic potential of CVC was evidenced by the ability to support growth of colony-forming units of hematopoietic progenitor cells from human CD34 ⁺ umbilical cord blood cells for a period of 5 weeks. Adipogenic potential was not observed. CVC were immunopositive to antigens to CD29 and CD44 but not to CD14 or CD45, consistent with other mesenchymal stem cells. CVC retained multipotentiality despite passaging and expansion through more than 20 to 25 population triplings, indicating a capacity for self-renewal. Conclusions— These results suggest that the artery wall contains cells that have the potential for multiple lineages similar to mesenchymal stem cells but with a unique differentiation repertoire.

Objectives— Osteoporosis is associated epidemiologically with atherosclerosis and hyperlipidemia. We previously found that atherogenic lipids regulate bone formation. To determine whether hyperlipidemia also affects bone resorption, we compared osteoclastogenesis in marrow preosteoclasts derived from hyperlipidemic versus control mice. Methods— Nonadherent marrow cells from low-density lipoprotein receptor−/− (LDLR−/−)and C57BL/6J mice were cultured with M-CSF and ligand for receptor activator of nuclear factor-kappaB (RANKL). Functional osteoclastic activity, measured as number of resorption pits, was significantly greater in 12-month-old LDLR−/−. Similar results were obtained in 5- and 10-month-old LDLR−/− versus C57BL/6J mice on a high-fat diet. Osteoclastic differentiation, indicated by tartrate resistant acid phosphatase (TRAP) activity, was significantly greater in the 12-month-old LDLR−/−, and there was a trend toward increased TRAP activity in LDLR−/− on a high-fat diet, at ages 5 and 10 months. Osteoclastic parameters correlated with total serum lipoproteins with a possible threshold effect. Osteoporotic human cortical bone stained positive for lipids in the perivascular space of Haversian canals by oil red O. The presence of lipid hydroperoxides was detected in bone marrow from hyperlipidemic mice. Conclusions— Hyperlipidemia may contribute to osteoporosis via increased osteoclastic bone resorption.

Hyperlipidemia increases the risk for generation of lipid oxidation products, which accumulate in the subendothelial spaces of vasculature and bone. Atherogenic high‐fat diets increase serum levels of oxidized lipids, which are known to attenuate osteogenesis in culture and to promote bone loss in mice. In this study, we investigated whether oxidized lipids affect bone regeneration and mechanical strength. Wild‐type (WT) and hyperlipidemic (Ldlr^−/−) mice were placed on a high‐fat (HF) diet for 13 weeks. Bilateral cranial defects were introduced on each side of the sagittal suture, and 5 weeks postsurgery on the respective diets, the repair/regeneration of cranial bones and mechanical properties of femoral bones were assessed. MicroCT and histological analyses demonstrated that bone regeneration was significantly impaired by the HF diet in WT and Ldlr^−/− mice. In femoral bone, cortical bone volume fraction (bone volume [BV]/tissue volume [TV]) was significantly reduced, whereas cortical porosity was increased by the HF diet in Ldlr^−/− but not in WT mice. Femoral bone strength and stiffness, measured by three‐point bending analysis, were significantly reduced by the HF diet in Ldlr^−/−, but not in WT mice. Serum analysis showed that the HF diet significantly increased levels of parathyroid hormone, tumor necrosis factor (TNF)‐α, calcium, and phosphorus, whereas it reduced procollagen type I N‐terminal propeptide, a serum marker of bone formation, in Ldlr^−/−, but not in WT mice. The serum level of carboxyl‐terminal collagen crosslinks, a marker for bone resorption, was also 1.7‐fold greater in Ldlr^−/− mice. These findings suggest that hyperlipidemia induces secondary hyperparathyroidism and impairs bone regeneration and mechanical strength. © 2012 American Society for Bone and Mineral Research

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-γ (PPAR-γ). Both p38-MAPK activator and PPAR-γ 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-γ pathways.

In hyperlipidemia, oxidized lipids accumulate in vascular tissues and trigger atherosclerosis. Such lipids also deposit in bone tissues, where they may promote osteoporosis. We found previously that oxidized lipids attenuate osteogenesis and that parathyroid hormone (PTH) bone anabolism is blunted in hyperlipidemic mice, suggesting that osteoporotic patients with hyperlipidemia may develop resistance to PTH therapy. To determine if oxidized lipids account for this PTH resistance, we blocked lipid oxidation products in hyperlipidemic mice with an ApoA-I mimetic peptide, D-4F, and the bone anabolic response to PTH treatment was assessed. Skeletally immature Ldlr^−/− mice were placed on a high-fat diet and treated with D-4F peptide and/or with intermittent PTH(1–34) injections. As expected, D-4F attenuated serum lipid oxidation products and tissue lipid deposition induced by the diet. Importantly, D-4F treatment attenuated the adverse effects of dietary hyperlipidemia on PTH anabolism by restoring micro–computed tomographic parameters of bone quality—cortical mineral content, area, and thickness. D-4F significantly reduced serum markers of bone resorption but not bone formation. PTH and D-4F, together but not separately, also promoted bone anabolism in an alternative model of hyperlipidemia, Apoe^−/− mice. In normolipemic mice, D-4F cotreatment did not further enhance the anabolic effects of PTH, indicating that the mechanism is through its effects on lipids. These findings suggest that oxidized lipids mediate hyperlipidemia-induced PTH resistance in bone through modulation of bone resorption. © 2011 American Society for Bone and Mineral Research.