Linda L. Demer

Atherosclerosis: basic mechanisms. Oxidation, inflammation, and genetics.

The clinical events resulting from atherosclerosis are directly related to the oxidation of lipids in LDLs that become trapped in the extracellular matrix of the subendothelial space. These oxidized lipids activate an NF kappa B-like transcription factor and induce the expression of genes containing NF kappa B binding sites. The protein products of these genes initiate an inflammatory response that initially leads to the development of the fatty streak. The progression of the lesion is associated with the activation of genes that induce arterial calcification, which changes the mechanical characteristics of the artery wall and predisposes to plaque rupture at sites of monocytic infiltration. Plaque rupture exposes the flowing blood to tissue factor in the lesion, and this induces thrombosis, which is the proximate cause of the clinical event. There appear to be potent genetically determined systems for preventing lipid oxidation, inactivating biologically important oxidized lipids, and/or modulating the inflammatory response to oxidized lipids that may explain the differing susceptibility of individuals and populations to the development of atherosclerosis. Enzymes associated with HDL may play an important role in protecting against lipid oxidation in the artery wall and may account in part for the inverse relation between HDL and risk for atherosclerotic clinical events.

Bone morphogenetic protein expression in human atherosclerotic lesions.

Artery wall calcification associated with atherosclerosis frequently contains fully formed bone tissue including marrow. The cellular origin is not known. In this study, bone morphogenetic protein-2a, a potent factor for osteoblastic differentiation, was found to be expressed in calcified human atherosclerotic plaque. In addition, cells cultured from the aortic wall formed calcified nodules similar to those found in bone cell cultures and expressed bone morphogenetic protein-2a with prolonged culture. The predominant cells in these nodules had immunocytochemical features characteristic of microvascular pericytes that are capable of osteoblastic differentiation. Pericyte-like cells were also found by immunohistochemistry in the intima of bovine and human aorta. These findings suggest that arterial calcification is a regulated process similar to bone formation, possibly mediated by pericyte-like cells.

The Yin and Yang of Oxidation in the Development of the Fatty Streak A Review Based on the 1994 George Lyman Duff Memorial Lecture

Recent data support the hypothesis that the fatty streak develops in response to specific phospholipids contained in LDL that become trapped in the artery wall and become oxidized as a result of exposure to the oxidative waste of the artery wall cells. The antioxidants present within both LDL and the microenvironments in which LDL is trapped function to prevent the formation of these biologically active, oxidized lipids. Enzymes associated with LDL and HDL (eg, platelet activating factor acetylhydrolase) or with HDL alone (eg, paraoxonase) destroy these biologically active lipids. The regulation and expression of these enzymes are determined genetically and are also significantly modified by environmental influences, including the acute-phase response or an atherogenic diet. The balance of these multiple factors leads to an induction or suppression of the inflammatory response in the artery wall and determines the clinical course.

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.

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.

Active Serum Vitamin D Levels Are Inversely Correlated With Coronary Calcification

BACKGROUND Arterial calcification is a common feature of atherosclerosis, occurring in >90% of angiographically significant lesions. Recent evidence from this and other studies suggests that development of atherosclerotic calcification is similar to osteogenesis; thus, we undertook the current investigation on the potential role of osteoregulatory factors in arterial calcification. METHODS AND RESULTS We studied two human populations (173 subjects) at high and moderate risk for coronary heart disease and assessed them for associations between vascular calcification and serum levels of the osteoregulatory molecules osteocalcin, parathyroid hormone, and 1alpha,25-dihydroxyvitamin D3 (1,25-vitamin D). Our results revealed that 1,25-vitamin D levels are inversely correlated with the extent of vascular calcification in both groups. No correlations were found between extent of calcification and levels of osteocalcin or parathyroid hormone. CONCLUSIONS These data suggest a possible role for vitamin D in the development of vascular calcification. Vitamin D is also known to be important in bone mineralization; thus, 1,25-vitamin D may be one factor to explain the long observed association between osteoporosis and vascular calcification.

Expression and Function of PPARγ in Rat and Human Vascular Smooth Muscle Cells

Background—Peroxisome proliferator–activated receptor-γ (PPARγ) is activated by fatty acids, eicosanoids, and insulin-sensitizing thiazolidinediones (TZDs). The TZD troglitazone (TRO) inhibits vascular smooth muscle cell (VSMC) proliferation and migration in vitro and in postinjury intimal hyperplasia. Methods and Results—Rat and human VSMCs express mRNA and nuclear receptors for PPARγ1. Three PPARγ ligands, the TZDs TRO and rosiglitazone and the prostanoid 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2), all inhibited VSMC proliferation and migration. PPARγ is upregulated in rat neointima at 7 days and 14 days after balloon injury and is also present in early human atheroma and precursor lesions. Conclusions—Pharmacological activation of PPARγ expressed in VSMCs inhibits their proliferation and migration, potentially limiting restenosis and atherosclerosis. These receptors are upregulated during vascular injury.

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.