Yin Tintut

Vascular Calcification: Mechanisms and Clinical Ramifications

Vascular calcification, long thought to result from passive degeneration, involves a complex, regulated process of biomineralization resembling osteogenesis. Evidence indicates that proteins controlling bone mineralization are also involved in the regulation of vascular calcification. Artery wall cells grown in culture are induced to become osteogenic by inflammatory and atherogenic stimuli. Furthermore, osteoclast-like cells are found in calcified atherosclerotic plaques, and active resorption of ectopic vascular calcification has been demonstrated. In general, soft tissue calcification arises in areas of chronic inflammation, possibly functioning as a barrier limiting the spread of the inflammatory stimulus. Atherosclerotic calcification may be one example of this process, in which oxidized lipids are the inflammatory stimulus. Calcification is widely used as a clinical indicator of atherosclerosis. It progresses nonlinearly with time, following a sigmoid-shaped curve. The relationship between calcification and clinical events likely relates to mechanical instability introduced by calcified plaque at its interface with softer, noncalcified plaque. In general, as calcification proceeds, interface surface area increases initially, but eventually decreases as plaques coalesce. This phenomenon may account for reports of less calcification in unstable plaque. Vascular calcification is exacerbated in certain clinical entities, including diabetes, menopause, and osteoporosis. Mechanisms linking them must be considered in clinical decisions. For example, treatments for osteoporosis may have unanticipated effects on vascular calcification; the converse also applies. Further understanding of processes governing vascular calcification may yield new therapeutic options for vascular disease.

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.

Vascular Calcification Pathobiology of a Multifaceted Disease

Clinically, vascular calcification is now accepted as a valuable predictor of coronary heart disease.151 Achieving control over this process requires understanding mechanisms in the context of a tightly-controlled regulatory network, with multiple, nested feedback loops and cross-talk between organ systems, in the realm of control theory. Thus, treatments for osteoporosis such as calcitriol, estradiol, bisphosphonates, calcium supplements, and intermittent parathyroid hormone are likely to affect vascular calcification, and, conversely, many treatments for cardiovascular disease such as statins, antioxidants, hormone replacement therapy, ACE inhibitors, fish oils, and calcium channel blockers may affect bone health. As we develop and use treatments for cardiovascular and skeletal diseases, we must give serious consideration to the implications for the organ at the other end of the bone-vascular axis.

Tumor Necrosis Factor-α Promotes In Vitro Calcification of Vascular Cells via the cAMP Pathway

BackgroundVascular calcification is an ectopic calcification that commonly occurs in atherosclerosis. Because tumor necrosis factor-&agr; (TNF-&agr;), a pleiotropic cytokine found in atherosclerotic lesions, is also a regulator of bone formation, we investigated the role of TNF-&agr; in in vitro vascular calcification. Methods and ResultsA cloned subpopulation of bovine aortic smooth muscle cells previously shown capable of osteoblastic differentiation was treated with TNF-&agr;, and osteoblastic differentiation and mineralization were assessed. Treatment of vascular cells with TNF-&agr; 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-&agr; 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-&agr;. Consistent with this, the intracellular cAMP level was elevated after TNF-&agr; treatment. Electrophoretic mobility shift assay demonstrated that TNF-&agr; enhanced DNA binding of osteoblast specific factor (Osf2), AP1, and CREB, transcription factors that are important for osteoblastic differentiation. ConclusionsThese results suggest that TNF-&agr; enhances in vitro vascular calcification by promoting osteoblastic differentiation of vascular cells through the cAMP pathway.

Multilineage Potential of Cells From the Artery Wall

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-&agr; 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.

Regulatory mechanisms in vascular calcification

In the past decade, the prevalence, significance, and regulatory mechanisms of vascular calcification have gained increasing recognition. Over a century ago, pathologists recognized atherosclerotic calcification as a form of extraskeletal ossification. Studies are now identifying the mechanism of this remarkable process as a recapitulation of embryonic endochondral and membranous ossification through phenotypic plasticity of vascular cells that function as adult mesenchymal stem cells. These embryonic developmental programs, involving bone morphogenetic proteins and potent osteochondrogenic transcription factors, are triggered and modulated by a variety of inflammatory, metabolic, and genetic disorders, particularly hyperlipidemia, chronic kidney disease, diabetes, hyperparathyroidism, and osteoporosis. They are also triggered by loss of powerful inhibitors, such as fetuin A, matrix Gla protein, and pyrophosphate, which ordinarily restrict biomineralization to skeletal bone. Teleologically, soft-tissue calcification might serve to create a wall of bone to sequester noxious foci such as chronic infections, parasites, and foreign bodies. This Review focuses on atherosclerotic and medial calcification. The capacity of the vasculature to produce mineral in culture and to produce de novo, vascularized, trabecular bone and cartilage tissue, even in patients with osteoporosis, should intrigue investigators in tissue engineering and regenerative biology.

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.

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) γ agonist thiazolidinediones, BRL49653 and ciglitizone. Based on promoter‐reporter construct experiments, MM‐LDL may be acting in part through activating PPARα. 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.”

Osteoprotegerin Inhibits Vascular Calcification Without Affecting Atherosclerosis in ldlr(−/−) Mice

Background— The role of osteoprotegerin in vascular disease is unclear. Recent observational studies show that serum osteoprotegerin levels are associated with the severity and progression of coronary artery disease, atherosclerosis, and vascular calcification in patients. However, genetic and treatment studies in mice suggest that osteoprotegerin may protect against vascular calcification. Methods and Results— To test whether osteoprotegerin induces or prevents vascular disease, we treated atherogenic diet–fed ldlr(−/−) mice with recombinant osteoprotegerin (Fc-OPG) or vehicle for 5 months. Vehicle-treated mice developed significant, progressive atherosclerosis with increased plasma osteoprotegerin levels, consistent with observational studies, and ≈15% of these atherosclerotic lesions developed calcified cartilage-like metaplasia. Treatment with Fc-OPG significantly reduced the calcified lesion area without affecting atherosclerotic lesion size or number, vascular cytokines, or plasma cholesterol levels. Treatment also significantly reduced tissue levels of aortic osteocalcin, a marker of mineralization. Conclusions— These data support a role for osteoprotegerin in the vasculature as an inhibitor of calcification and a marker, rather than a mediator, of atherosclerosis.

Mesenchymal Stem Cells and the Artery Wall

The presence of ectopic tissue in the diseased artery wall is evidence for the presence of multipotential stem cells in the vasculature. Mesenchymal stem cells were first identified in the marrow stroma, and they differentiate along multiple lineages giving rise to cartilage, bone, fat, muscle, and vascular tissue in vitro and in vivo. Transplantation studies show that marrow-derived mesenchymal stem cells appear to enter the circulation and engraft other tissues, including the artery wall, at sites of injury. Recent evidence indicates that mesenchymal stem cells are also present in normal artery wall and microvessels and that they also may enter the circulation, contributing to the population of circulating progenitor cells and engrafting other tissues. Thus, the artery wall is not only a destination but also a source of progenitor cells that have regenerative potential. Although potential artifacts, such as fusion, need to be taken into consideration, these new developments in vascular biology open important therapeutic avenues. A greater understanding of how mesenchymal stem cells from the bone marrow or artery wall bring about vascular regeneration and repair may lead to novel cell-based treatments for cardiovascular disease.