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Molecular, Endocrine, and Genetic Mechanisms of Arterial Calcification

Atherosclerosis Research Center and Burns and Allen Research Institute (T.M.D., P.K.S., T.B.R.), Division of Cardiology, Department of Medicine, Cedars-Sinai Medical Center and David Geffen School of Medicine at the University of California Los Angeles (UCLA), Los Angeles, California 90048; Division of Endocrinology, Diabetes, Metabolism, and Nutrition (L.A.F.), Department of Internal Medicine, Mayo Clinic and Mayo Foundation, Rochester, Minnesota 55905; Division of Endocrinology and Metabolism (D.I.), Department of Medicine and Bioregulatory Sciences, University of Tokushima Graduate School of Medicine, Tokushima 770-8503, Japan; Department of Pathology and Laboratory Medicine (J.-H.Q., M.C.F.), David Geffen School of Medicine at UCLA, Los Angeles, California 90095; and Division of Cardiology (R.C.D.), Department of Medicine, Harbor-UCLA Research and Education Institute, Torrance, California 90512

Correspondence: Address all correspondence and requests for reprints to: Tripathi B. Rajavashisth, Ph.D., Atherosclerosis Research Center, Davis Research Building, Room 1062, Division of Cardiology, Department of Medicine, Cedars-Sinai Medical Center and David Geffen School of Medicine at University of California, Los Angeles, 8700 Beverly Boulevard, Los Angeles, California 90048-1865. E-mail: rajavashisth{at}cshs.org

	Abstract

Top Abstract I. Introduction: Clinical... II. Types of Arterial... III. Molecular Mechanisms of... IV. Endocrine, Pharmacological,... V. Genetic Determinants of... VI. Conclusions References

 
Pathologists have recognized arterial calcification for over a century. Recent years have witnessed a strong resurgence of interest in atherosclerotic plaque calcification because it: 1) can be easily detected noninvasively; 2) closely correlates with the amount of atherosclerotic plaque; 3) serves as a surrogate measure for atherosclerosis, allowing preclinical detection of the disease; and 4) is associated with heightened risk of adverse cardiovascular events. There are two major types of calcification in arteries: calcification of the media tunica layer (sometimes called Mönckeberg’s sclerosis), and calcification within subdomains of atherosclerotic plaque within the intimal layer of the artery. There are important similarities and differences between these two entities. Of particular interest are increasing parallels between cellular and molecular features of arterial calcification and bone biology, and this has led to accelerating interest in understanding how and why bone-like mineral deposits may form in arteries. Here, we review the two major pathological types of arterial calcification, the proposed models of calcification, and endocrine and genetic determinants that affect arterial calcification. In addition, we highlight areas requiring further investigation.

I. Introduction: Clinical Perspective

II. Types of Arterial Calcification

A. Medial calcification

B. Intimal calcification

III. Molecular Mechanisms of Arterial Calcification

A. Molecular and genetic mechanisms of bone formation

B. The active model of arterial calcification

C. The potential role of OLCs in arterial calcification

D. The passive physicochemical model of arterial calcification

E. A unified model

IV. Endocrine, Pharmacological, and Lipid Influences on Arterial Calcification

A. Parathyroid hormone and vitamin D

B. Estrogen

C. Pharmaceutical agents that regulate calcification

D. Role of lipids

V. Genetic Determinants of Arterial Calcification

A. Angiotensin-converting enzyme

B. Apolipoprotein E

C. E-selectin

D. Matrix metalloproteinase-3 (MMP-3)

E. Matrix Gla protein

F. CC chemokine receptor 2

G. Estrogen receptor-

H. Ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1)

I. Epoxide hydrolase

J. Osteoprotegerin

K. Other human genetic disorders and arterial calcification

VI. Conclusions

	I. Introduction: Clinical Perspective

Top Abstract I. Introduction: Clinical... II. Types of Arterial... III. Molecular Mechanisms of... IV. Endocrine, Pharmacological,... V. Genetic Determinants of... VI. Conclusions References

 
DESPITE TREMENDOUS ADVANCES in our understanding and treatment of cardiovascular disorders, coronary heart disease (CHD) now accounts for most death and disability in developed countries, exacts a financial toll approaching $500 million annually in the United States alone (1, 2), and will soon eclipse infection as the major cause of all morbidity and mortality (3, 4). Yet the presence of atherosclerotic plaque—the pathobiological substrate upon which the overwhelming majority of CHD events occurs—does not of itself lead irrevocably to poor outcomes (5). Atherosclerosis is characterized by chronic arterial inflammation instigated and exacerbated by disordered lipid metabolism and other well-characterized risk factors (6, 7, 8, 9). Large postmortem studies have revealed that atherosclerosis begins surprisingly early and is ubiquitous in middle-aged and older adults (10, 11) (Fig. 1), yet epidemiological studies have paradoxically shown that despite the very high prevalence of atherosclerosis in adults, most will never suffer a clinical event from the disease and will ultimately die of other causes (5, 12, 13). The resolution of this paradox came from seminal investigations that elegantly established that the majority of devastating CHD events such as myocardial infarction are directly precipitated by rupture or erosion of unstable plaque, and it is the composition rather than the amount or size of plaque that critically determines instability (14, 15, 16). These startling findings lead to the disquieting realization that individuals with fairly extensive atherosclerosis may nonetheless lead normal event-free lives if their disease remains structurally quiescent, but those with relatively little atherosclerosis can and do suffer devastating CHD events if they have plaque that becomes vulnerable to rupture. Accordingly, the focus of vascular biologists and clinical scientists has now shifted toward understanding how plaque composition is determined and why and how it becomes vulnerable.

View larger version (99K): [in this window] [in a new window]   FIG. 1. The spectrum of atherosclerosis from the cradle to the grave. Fatty streaks, neointimal thickening, and raised dots or plaques on the arterial surface can appear during infancy. A, Scanning electron micrograph of an intact endothelial layer over a raised intimal lesion. B, Photograph of raised fatty dots on the surface of the aorta from a 5-yr-old child obtained at autopsy. Plaques grow from within the artery, but because the artery may undergo progressive compensatory dilatation (C), blood flow may remain unimpeded unless the majority of the cross-sectional area becomes diseased and the artery can no longer dilate further. The lesion shown in panel C (stained with oil red O, lipid appears red) appeared normal on angiography despite significant plaque. Often without warning, a plaque surface may tear or become eroded, exposing thrombogenic arterial components to flowing blood. D, A large thrombus adhering to the luminal arterial surface that was due to endothelial erosion or tearing of the plaque cap. Note that the plaque is intact and the degree of stenosis is only mild. Such plaques are not severe enough to compromise blood flow and are therefore typically asymptomatic, but they can nonetheless be lethal. Small blood vessels within the plaque may also hemorrhage into an otherwise structurally intact plaque. If intraplaque bleeding is extensive (E), the luminal diameter of the artery may be compromised, causing ischemia. F, Complete thrombotic occlusion has occurred and is associated with a large, lipid-filled lesion. G, A similar plaque. Again, total thrombotic occlusion of the artery has occurred (green arrow). Note that there is also a small intraplaque hemorrhage (blue arrow). The plaque cap appears to be intact, suggesting that thrombotic occlusion of this artery occurred by a mechanism that did not involve outright structural failure of the plaque cap. In such types of thrombosis, platelet-rich thrombi may form on damaged mural surfaces and then either resolve spontaneously or develop into much larger clots that occlude the lumen of the artery. H, A plaque whose overlying cap has obviously ruptured (white arrows). When arterial blood comes into contact with subendothelial tissues in the artery wall, thrombosis very rapidly forms, and it may fill the plaque and continue to propagate into the lumen of the artery, causing an acute coronary event such as unstable angina, myocardial infarction, or sudden death. Note that this plaque also is associated with only mild obstruction of the arterial lumen. Together, these images underscore a quintet of critical concepts: 1) atherosclerosis emerges early in life; 2) its progression is closely linked to the dynamics of plaque structure; 3) its natural history tends to be episodic and nonlinear with respect to time; 4) outcomes are not closely related to the severity of the disease, as reflected by plaque size or degree of stenotic obstruction; and 5) rapid worsening and precipitation of devastating events may suddenly occur without warning, even when the amount of disease is minimal and undetectable by anatomic (e.g., coronary angiography) or physiological (e.g., exercise stress testing) diagnostic tests. [Panels A–F, H reproduced with permission from M. J. Davies (713 ); panel G reproduced with permission from H. C. Stary (11 ).]

One highly prevalent component of atherosclerotic plaque is calcium mineral deposits. Arterial calcification has attracted intense interest because it can be readily quantified noninvasively with radiographic imaging techniques (Fig. 2), and clinical studies have shown that high amounts of coronary artery calcification predict a heightened risk of myocardial infarction and sudden coronary death (20, 21, 22, 23, 24). At least in the coronary arteries, calcification seems to invariably indicate the presence of plaque, but if calcification is not present, it cannot be reliably concluded that plaque is absent (25, 26). Like atherosclerosis itself, arterial calcification begins quite early in life and increases with age at a rate that is roughly commensurate with the rate that atherosclerosis develops. By the seventh or eighth decade, the prevalence of arterial calcification, like atherosclerosis, is virtually universal, but may be present in amounts that vary widely among individuals. Calcification of plaques may precede clinical manifestations of plaque instability by many years, leading to the notion that detection of "subclinical" atherosclerosis by the use of a surrogate measure—arterial calcification—might allow accurate prospective identification of those destined to suffer clinical syndromes caused by atherosclerosis, while at the same time delineating those who are very unlikely to suffer an event. The clinical issues and pertinent evidence are controversial, far from straightforward, and beyond the scope of this review. For more detailed discussion, the reader is referred to recent expert consensus guidelines issued jointly by the American Heart Association and the American College of Cardiology (21), metaanalyses of clinical data (22, 24), and a number of commentaries (27, 28, 29, 30, 31). Here, we will limit our focus to potential molecular, endocrine, and genetic mechanisms mediating calcification in atherosclerotic plaque.

View larger version (100K): [in this window] [in a new window]   FIG. 2. Arterial calcification as detected by various imaging modalities. A–E, Intravascular ultrasound (IVUS) images of human coronary arteries. A normal artery (A) and an atherosclerotic artery with no calcification (B) are shown. C, Moderate calcification (arrows) in association with a large atherosclerotic plaque (dashed red region). D and E, Heavily calcified plaque, which almost completely encircles the artery in E (arrows). F, A radiograph of a heart taken at autopsy. The proximal sections of all three major coronary arteries are outlined by calcification. G–J, More modern radiographic imaging methods include CT. I and J, Three-dimensional reconstruction of images is possible. K–P, In vitro radiographic analytic imaging methods recently developed include high resolution synchrotron radiation x-ray microcomputed tomography. This method also allows three-dimensional image reconstruction and rendering. Calcification in a plaque from a human artery (K) and a saphenous vein bypass graft (L) are shown. K, Arrows indicate the arterial wall (top arrow), calcium deposits (middle arrow), and the sample holder (bottom arrow). M–P, Variations in volumetric three-dimensional rendering methods with synchrotron tomographic images. Arrows indicate the vessel lumen in M and O and calcium deposits in N and P. [Panels A–D courtesy of Dr. Steven L. Goldberg, University of Washington, Seattle, WA; panel E reproduced with permission from S. E. Nissen et al. (714 ); panel F reproduced with permission from M. J. Davies (713 ); panels G–J courtesy of Dr. Matthew J. Budoff, Harbor-UCLA Medical Center; panels K and L reproduced with permission from H. Jin et al.: Phys Med Biol 47:4345–4356, 2002 (715 ); panels M and N courtesy of Dr. Richard L. Kurtz, Louisiana State University.]

	II. Types of Arterial Calcification

Top Abstract I. Introduction: Clinical... II. Types of Arterial... III. Molecular Mechanisms of... IV. Endocrine, Pharmacological,... V. Genetic Determinants of... VI. Conclusions References

View larger version (87K): [in this window] [in a new window]   FIG. 3. Histological examples of human arterial calcification. A, Arteries may exhibit concentric medial arterial calcification, which can occur in arteries free from atherosclerotic plaque. In this specimen, obtained during iliac artery surgery from a world-class athlete, there is concentric medial hypertrophy with medial calcification and little or no intima (Verhoeff-van-Gieson stain). Medial calcification may involve only elastic fibers or may sometimes develop into large calcific deposits. B, Calcification (black) in the media (red arrow) and in an intimal plaque (black arrow) in an artery from a patient with renal disease. The boxed area in panel B is magnified (x40) in panel C (I, intima; P, plaque; M, media; MacNeal’s tetrachrome staining). D and E, The human arterial specimen shown in panel D (with boxed area enlarged in E) demonstrates mineralization of medial elastic fibers and areas of large calcium deposits in the absence of significant intimal plaque. Calcification in atherosclerotic human arteries is usually localized in the deeper aspect of the intimal layer adjacent to the media. F–H, Typical well-developed calcification within intimal human plaques. Special embedding using methylmethacrylate allows sectioning without initial decalcification and reveals a fine, diffuse pattern of intimal calcification (G, arrow) in addition to large focal calcium deposits (teal color). It is not known whether diffuse calcification eventually becomes more organized into focal deposits. Goldner’s-Masson trichrome staining (F and G) reveals calcification that is not apparent on a section contiguous to panel F using hematoxylin and eosin alone (H). I and J, Circumferential calcification, largely intimal, can be seen in panel I, whereas more focal calcium deposits are seen in panel J (both human arteries stained with von Kossa; calcium is black). [Panel A reproduced with permission from C. A. Kral et al.: J Vasc Surg 36:565–570, 2002 (716 ); panels B and C reproduced with permission from S. M. Moe et al.: Kidney Int 63:1003–1011, 2003 (224 ).]

View larger version (107K): [in this window] [in a new window]   FIG. 4. Microscopic features of human atherosclerotic arterial calcification. A, Calcium in a lesion from a coronary artery of a 35-yr-old female. Small calcium mineral aggregates (blue arrows) are scattered throughout the lipid core, and macrophage foam cells (yellow arrow) cluster in an area of the core facing the lumen. B, A detail of this section at higher magnification. Calcium particles intermingle with cholesterol crystals and other lipids comprising the core of the lesion. C, Calcification (yellow arrows) and fibrosis (pink region denoted by asterisk) at the base of the lipid core of a lesion in the abdominal aorta. D, When lipid cores regress, residual areas of calcification may persist. The well-defined elastic lamina (arrows) separates a thin media and the adventitia (lower part of field) from the lesion. The subregion of the lesion adjacent to the elastic lamina consists of aggregates of calcium particles (denoted by asterisk) seen mostly as irregular empty spaces because of decalcification before sectioning. Residual extracellular lipid and cholesterol crystals can be seen in the midregion of the intimal plaque, with overlying SMCs and collagen. E, Focal calcification (basophilic blue-purple aggregates) associated with a macrophage-rich lipid core. Individual SMCs may become calcified as well. F and G, SMC containing variable amounts of calcium aggregates, some of which may represent calcified intracellular organelles. Calcium aggregates can also be seen in the intercellular space in panel G. Calcification in human plaques may form in focal intracellular and intercellular regions (as in F and G) but may also appear as a "wavefront" that produces plate-like circumferential intimal calcification. H, Mild mineralization of medial elastic lamellae (Mönckeberg’s sclerosis) in a human artery. von Kossa staining (brown-black) localizes only to elastic fibers and the internal elastic lamina (arrows). No staining is seen in the intima (red "In"). Media is denoted by a red "m." I, Advanced medial calcification (red arrow), with concomitant intimal calcification (green arrow). J, Artery exhibiting amorphous medial calcification (blue arrows), intimal (In) calcification (black arrows), and a region of bone (red arrow). Osteocytes can be seen (small nonstaining holes) within bone. K, Calcification (von Kossa, dark brown-black) within -SMC actin-stained media (small arrowheads) (m, media; Ad, adventitia; Ca, calcification; arrows indicate mineralized internal elastic lamina). L, Staining by the macrophage marker CD68 is seen in the adventitia but not in the media or in the vicinity of areas of calcification. A–D, F, and G, Toluidine blue and basic fuchsin stain; E, hematoxylin and eosin; H–L, von Kossa stain. [Panels A–D, F and G reproduced with permission from H. C. Stary (11 ); panel E reproduced with permission from M. J. Davies (713 ); panels H–L reproduced with permission from C. M. Shanahan et al.: Circulation 100:2168–2176, 1999 (126 ).]

View larger version (79K): [in this window] [in a new window]   FIG. 5. Bone formation in human arteries. A–D, Examples of bone formation with mineralized trabeculae in arteries. B, Mature lamellar bone (white B) in a carotid artery plaque (red P), with a focus of calcification (red C). Osteocytes in lacunae (arrowheads) and an osteoclast in a resorption space (red arrow) can be seen. Mast cells and mononuclear cells (arrow) are seen in association with bone (C). D, Calcification (dark-colored areas within the intensely red-stained regions) integrated within mineralized bone tissue (intense red staining; hemotoxylin and eosin staining). E, Staining with von Kossa (black) and counterstaining with Safranin O reveals osteocytes and adjacent cells. F–N, High-magnification details of arterial bone tissue. F, Marrow-like tissue containing lymphocytes, mast cells, monocytes, and blood vessels within a mineralized region. Macrophages (brown, stained with HAM56 in panel G; and CD68, black arrows in panels I and N), SMCs (H), giant cells (K and N, arrows), multinucleated TRAP-positive osteoclasts (I, green arrow; J, black arrow) are shown in close association with arterial bone tissue. CD68-positive macrophages and giant cells (L and M, arrows) are seen adjacent to mineralized regions (L and M, asterisks), colocalized with BMP-6 expression (M). O–R, Atherosclerotic arteries demonstrating calcification(O, green arrows), cartilaginous metaplasia (O, black arrows), chondrocytes and chondroid tissue (Q and R, dark blue/purple regions), osteoblasts (P, black arrows) on the surface of an osseous region (P, asterisk), and multinuclear OLCs (P, green arrows). Thrombus (T) can be seen in the lumen of the artery shown in panel O. [Panels A, C–M reproduced with permission from M. Jeziorska et al.: Virchows Arch 433:559–565, 1998 (238 ); panel B reproduced with permission from J. L. Hunt et al.: Stroke 33:1214–1219, 2002 (237 ); panel N reproduced with permission from M. Jeziorska et al.: J Pathol 185:10–17, 1998 (259 ).]

Medial calcification can be produced experimentally by a variety of treatments, including administration of vitamin D in rats (50, 51, 52, 53, 54, 55), pigs (56), and rabbits (57); vitamin D plus nicotine with or without supplemental cholesterol in rats (58, 59, 60, 61) and rabbits (62, 63); vitamin D with magnesium deficiency in pigs (64) and rabbits with chronic renal failure induced by renal cauterization and contralateral nephrectomy (65); 1- hydroxycholecalciferol with or without parathyroidectomy in rats rendered uremic by subtotal kidney resection (66); warfarin with (51, 67, 68, 69, 70, 71) or without (70) vitamin K supplementation in rats; and vitamin D, warfarin, and vitamin K in rats (71, 72) (Fig. 6).

View larger version (26K): [in this window] [in a new window]   FIG. 6. Outline of the major genetic, molecular, cellular, and endocrine mediators of bone remodeling. In developing embryos, embryonic stem cells beget more restricted pluripotent progenitors that play important roles in tissue repair and remodeling processes in the adult. Mesenchymal and hematopoietic stem cells give rise to increasingly specialized cells that mediate bone formation and degradation, respectively. Mesenchymal stem cells can differentiate further into cells of the adipocyte, myoblast, and chondrocyte lineages, depending on genetic and morphogenetic programs and cues that are not well understood. Endochondral bone formation involves a cartilage intermediate. Mesenchymal progenitors become progressively committed to the chondrocytic lineage, form increasingly ordered condensations, proliferate, differentiate, and begin elaborating matrix elements consisting largely of collagens and aggrecan. At around this time, chondrocytes begin to hypertrophy and enter apoptosis. Simultaneously, neovascularization into the mesenchymal tissues develops, and preosteoblast cells that have differentiated from mesenchymal progenitors migrate into the region occupied by the now dying chondrocytes. There, they develop into terminally differentiated osteoblasts and begin mineralization of the matrix template left behind by the chondrocytes. Meanwhile, hematopoietic progenitors give rise to mononuclear lineages destined to become monocytes, macrophages, and osteoclasts. Cells capable of producing each of these exist in bone marrow, and can also be found in the circulating mononuclear fraction in blood. Proper development and function of osteoclasts critically depend upon the cytokines CSF-1 and RANKL, which signal via their cognate cell-surface receptors (c-fms and RANK, respectively). These are produced by osteoblasts/stromal cells, as well as by various circulating cells. In bone, development of osteoclasts appears to interact closely with nearby osteoblasts/stromal cells. Giant multinucleated cells appear to arise by fusion of mononuclear cells. Bone degradation involves the formation of a sealed zone beneath the osteoclast via adhesion molecules, particularly vß3 integrin. Mineral degradation requires secretion of powerful proteases, such as cat K, and a marked decrease in pH under the osteoclast, accomplished primarily by proton-pumping enzymes such as ATP6i. The expression of TRAP, CA-II, cat K, CTR, ATP6i, and vß3 integrin in giant multinucleated cells defines mature, fully differentiated osteoclasts fully capable of mineral resorption. Bone remodeling also involves a coordination or coupling of the activities of osteoblasts and osteoclasts such that there is normally little or no net change in bone mass, but precisely how this is accomplished is not well understood. The major transcription factors, hormones, and cytokines implicated in osteoclastogenesis, chondrogenesis, and osteoblastogenesis are shown. Proteases including members of the MMP family play important roles in remodeling tissues to facilitate endochondral bone formation. Intramembraneous bone formation occurs mainly in certain bones of the skull and does not require a cartilage intermediate to form before mineralization by osteoblasts. A number of the cell types, transcription factors, signaling molecules, growth factors, and hormones shown have been found in calcified areas of atherosclerotic plaque. Their potential role in mediating plaque formation is currently under investigation.

Numerous reports have documented the high prevalence and extent of medial calcification commonly seen in patients with diabetes mellitus (82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94). Medial calcification appears to be an indicator of the severity and/or duration of diabetes, because it is closely associated with many of the complications and sequelae of diabetes, particularly autonomic neuropathy (90, 92, 95, 96, 97). However, there also may be an independent association of medial calcification with nondiabetic neuropathies. For example, 6–8 yr after bilateral lumbar sympathectomy, Goebel and Fuessl (98) found that 93% of patients exhibited medial arterial calcification in both feet, and most of these patients were not diabetic. Twenty patients had no evidence of medial calcification before surgery, but seven who underwent bilateral sympathectomy exhibited medial calcification in both feet, and 11 who received unilateral sympathectomy developed medial calcification only on the side of the operation. In general, after unilateral sympathectomy, the incidence of calcified arteries on the same side as the operation was higher than that on the contralateral side (88 vs. 18%; P < 0.01). Diabetic patients appeared to have more extensive calcification than nondiabetic subjects, but the apparent differences between groups were not significant. Thus, sympathetic denervation can cause Mönckeberg’s sclerosis regardless of whether diabetes mellitus is also present or not (98). Young et al. (90) found that nonneuropathic diabetic patients and age-matched nondiabetic patients had a similar prevalence of medial arterial calcification, but on the other hand, they also found a moderate but significant correlation (r = 0.32) between medial calcification and duration of diabetes. Logistic regression showed that serum creatinine, vibration perception, and duration of diabetes predicted the probability of vascular calcification. However, several studies have shown that in patients with diabetes mellitus and/or renal dysfunction, quantification of coronary artery calcification is not an accurate marker of the severity of coronary atherosclerosis determined by angiography (99) and poorly predicts clinical outcomes (100). One potential explanation is that in such patients, increased amounts of medial calcification that is not associated with intimal plaque tend to weaken the relationship between calcification and atherosclerosis, and thereby significantly dilute predictive power. However, although it is clear that assessment of calcification in such patients is of little value, much remains to be learned about the specific and perhaps unique features of atherosclerosis in the setting of diabetes and renal dysfunction (80).

Genetic analyses on a large group of Pima Indians in the southwest United States also supports the notion that medial calcification may have determinants that are independent of diabetes. These Indians have been extensively studied (101, 102, 103, 104, 105, 106, 107, 108, 109, 110) because they have one of the world’s highest reported rates of obesity, insulin resistance, and type 2 diabetes (109, 111, 112, 113) but nevertheless exhibit relatively low risk for atherosclerosis and the cardiovascular complications that are highly prevalent in other diabetic populations (114, 115, 116). As might be anticipated, Pima Indians have a very high rate of medial arterial calcification. However, Narayan et al. (117) showed that offspring of parents with medial calcification were significantly more likely to have medial calcification, even after adjusting for the effects of age, sex, diabetes, serum cholesterol, and blood pressure. Offspring of one parent with medial calcification had an odds ratio (OR) of 3.3 for having calcification [95% confidence interval (CI), 1.5 to 7.6], whereas if both parents manifested medial calcification, the OR increased to 8.1 (95% CI, 3.4 to 18.8). Taken together, these studies are most consistent with the interpretation that medial artery calcification is strongly associated with diabetes, but in addition there is a genetic component that is independent of the determinants of diabetes and also of atherosclerotic disease.

In patients with diabetes, medial arterial calcification may be associated with increased risk of cardiovascular complications. For example, Lehto et al. (89) found that medial calcification was a strong independent predictor of total (risk factor-adjusted OR, 1.6; and 95% CI, 1.2 to 2.2), cardiovascular (OR, 1.6; 95% CI, 1.1 to 2.2), and CHD (OR, 1.5; 95% CI, 1.0 to 2.2) mortality. Others have similarly reported that medial calcification is associated with increased risk of future cardiovascular events in patients with diabetes (118). On the other hand, Maser et al. (119) studied 657 patients with insulin-dependent diabetes in the Pittsburgh Epidemiology of Diabetes Complications Study and did not find that medial arterial calcification was an independent predictor of elevated cardiovascular risk. The reason(s) for these discordant results are unclear. Edmonds (82) has suggested that the deleterious sequelae of medial calcification in patients with diabetes may be largely secondary to stiffening of arterial tone, increased systolic blood pressure, and impairment of endothelium-dependent relaxation (55), all of which would be expected to cause abnormal flow characteristics that may facilitate or synergize common complications of diabetes such as atherogenesis. How this might occur is unclear at this point, particularly in view of evidence that suggests that in patients without diabetes mellitus, medial arterial calcification may not predict increased cardiovascular risk. For example, Everhart et al. (120) found that nondiabetic subjects with medial arterial calcification did not have higher mortality rates than subjects without medial arterial calcification. A recent report by London et al. (121) assessed intimal carotid arterial calcification with carotid B-mode ultrasonography and peripheral medial calcification with soft-tissue x-rays of the pelvis and thigh in 202 patients on stable hemodialysis. Not surprisingly, intimal carotid calcification was more often seen in older patients with a prior history of cardiovascular disease, whereas peripheral medial calcification was typically seen in young and middle-aged patients without increased cardiovascular risk. Medial calcification was linked to duration of hemodialysis and calcium-phosphate electrolyte disorders. Both types of calcification were associated with all-cause and cardiovascular mortality upon follow-up, independent of risk factors. Patients with medial calcification survived longer. These findings must be interpreted with considerable caution, because both enrollment of subjects and interpretation of outcomes were susceptible to a number of sources of selection bias (122, 123, 124). Probably the most clearly interpretable result from this study was the association of metabolic disorders of calcium and phosphate metabolism with medial calcification. Other evidence also supports the conclusion that metabolic disorders can contribute importantly to medial calcification (125).

Studies of medial calcification typically have relied upon radiographic detection methods (such as plain x-ray films) that are very specific but have low sensitivity. Additional studies in both diabetic and nondiabetic cohorts using imaging modalities with higher sensitivity may help to better elucidate the prognostic significance of medial calcification. At this point, based on available data, it seems reasonable to tentatively conclude that medial calcification probably imparts higher cardiovascular risk in diabetic patients, but not in nondiabetic patients.

Several studies have reported differential expression of bone matrix proteins in medial calcification compared with normal arteries or to intimal calcification. For example, Shanahan et al. (126) compared mRNA and protein expression of bone matrix proteins in human peripheral arteries with and without medial calcification. Normal arteries expressed matrix Gla protein (MGP) and osteonectin in the media. In the medial layer of the arteries with medial calcification, there were decreased levels of expression of MGP and osteonectin, but increased expression of alkaline phosphatase, BSP, bone Gla protein (BGP, or osteocalcin), and collagen II compared with normal vessels. Medial calcification was noted to invariably occur directly adjacent to medial smooth muscle cells (SMCs) expressing typical SMC markers such as SMC actin, but was never seen in the vicinity of macrophages or lipid deposits (126).

Other studies have documented expression of MGP in the media closely associated with SMCs and elastic lamellae. Extensive medial calcification is seen in MGP–/– mice (127, 128), which has led to the suggestion that MGP may have a homeostatic role in chelating or sequestering Ca²⁺ ions, thereby inhibiting mineralization. In addition to MGP, other proteins can bind or chelate Ca²⁺ ions. However, genetic ablation of several of these, including OPN (129, 130), osteonectin (131, 132), and BGP (133), does not produce arterial calcification in mice. Interestingly, double knockout (OPN–/–; MGP–/–) mice exhibited accelerated arterial calcification compared with single knockout (OPN+/+; MGP–/–) mice (134). Recent studies by Bostrom and colleagues (135, 136, 137) suggest that MGP may inhibit chondrocyte development by acting as an extracellular inhibitor of bone morphogenetic proteins (BMPs). This may explain why numerous chondrocytes are observed in arteries of MGP knockout mice (127, 128).

B. Intimal calcification
The remainder of this review will focus on arterial calcification observed in the setting of atherosclerotic plaque. This type of calcification occurs almost exclusively in the intimal layer of the artery (25). Normal arteries demonstrate little or no intima (9, 138, 139). However, at sites of hemodynamic stress, fibromuscular proliferation, referred to as adaptive intimal thickening, does occur and is reported to be the site at which clinically significant atheroma develops in the coronary arteries (6, 7, 9, 138, 139) (Fig. 1). Calcification in the intima occurs in at least two distinct patterns (Figs. 3 and 4). First, there are clumps of mineralized areas that have been described as punctate; these may enlarge, and most often are noted in the basal regions of the intima adjacent to the media. Sometimes these may appear organized, and foci of calcification may undergo osseous metaplasia, which includes bone marrow as well as bone (Fig. 5). Hematopoietic marrow, osteoblast-like cells, chondrocyte-like cells, multinucleated osteoclast-like cells (OLCs), and perhaps even osteocytes may be present in association with proteins normally associated with bone metabolism and typically not expressed in normal arteries (140, 141).

Pathologists routinely process arterial specimens by an initial decalcification step, which is intended to remove enough calcium deposits to enable tissue sectioning with standard microtomes. Although this facilitates rapid tissue sample processing, it is likely that the amount of calcification observed will be underestimated. Innovative histochemical techniques performed by Fitzpatrick and colleagues (142, 143) that do not require a decalcification step have now established that there is a fine, diffuse pattern of calcification that commonly occurs in atherosclerotic plaque in addition to the punctate pattern (Fig. 4). This diffuse pattern has several important characteristics and implications. First, it may be observed in virtually all areas of the intima. Second, it is unlikely to be detected by radiographic or other imaging modalities, because the overall tissue density is nearly the same as that of adjacent tissue that does not exhibit any calcification at all. Third, the natural history of the diffuse pattern and its relationship to the punctate pattern is not known. It is possible that they are independent of one another, but it is also conceivable that the diffuse pattern may, at least in some cases, be an early stage of what eventually becomes the punctate pattern. Resolving these questions will be an important goal of future studies.

	III. Molecular Mechanisms of Arterial Calcification

Top Abstract I. Introduction: Clinical... II. Types of Arterial... III. Molecular Mechanisms of... IV. Endocrine, Pharmacological,... V. Genetic Determinants of... VI. Conclusions References

 
A number of studies have shown intriguing parallels between arterial calcification in atherosclerotic plaque and osteogenesis. Over a century ago, pathologists observed that fully formed bone tissue, including marrow, can form in atherosclerotic arteries (144, 145) (Fig. 5). More recently, cells, proteins, and cytokines known to be involved in new bone formation have been reported to be present in arteries, and there is increased expression of a number of bone-related proteins in atherosclerotic plaque, particularly in subdomains of plaque where calcification occurs (23, 141, 146, 147, 148). Because of these similarities, it has been proposed that the mechanism by which arterial calcification occurs is similar to the mechanism of new bone formation (141, 148, 149, 150, 151). Although the parallels are intriguing and suggestive, these data are either observational expression analyses of bone-related proteins in arteries or extrapolations based upon in vitro cell culture studies. Direct tests of possible mechanisms using in vivo models have not yet been reported.

Nevertheless, several models postulating mechanisms for the formation and/or inhibition of calcification have now been proposed (141). These are: 1) the active model; 2) the passive physicochemical model; and 3) the arterial OLC model. Each model will be described below, and all are uniquely predicated on specific principles of bone formation and/or resorption. These models are not necessarily mutually exclusive alternatives, but may be complementary. Widely accepted principles of both vascular and bone biology form the foundation for these models and will therefore be briefly reviewed.

A. Molecular and genetic mechanisms of bone formation
In vertebrates, the three-dimensional pattern of the endoskeleton is specified by complex genetic programs derived from, and related to, phylogenetically ancient signaling systems that direct embryogenesis and morphology (152, 153, 154). Osteoblasts are responsible for bone formation (155, 156). Osteoclasts degrade bone, and thus counterbalance the actions of osteoblasts on overall bone mass under homeostatic conditions (157, 158) (Fig. 6). These cell types are derived from mesenchymal and hematopoietic (mononuclear phagocyte) precursors, respectively. Bone is formed in one of two distinct ways. The major skeletal elements develop by endochondral ossification that involves a cartilage intermediate, but some bones, notably the craniofacial bones, are formed by intramembraneous ossification in which bone arises directly from mesenchymal cell condensates without a cartilaginous intermediate (159, 160). Chondrocytes are the third major cell type involved in bone formation, are derived from mesenchymal cells, function to generate an initial cartilage template upon which endochondral bone formation including mineral deposition may occur, and develop in response to specific genetic programs (161, 162). Chondrocytes and osteoblasts appear to be derived from a common progenitor cell (159). Chondrocytes deposit a cartilagespecific extracellular matrix, proliferate, become hypertrophic during maturation, and undergo apoptosis. Surrounding mesenchymal cells differentiate into osteoblasts and invade the zones occupied by hypertrophic and dying chondrocytes. Simultaneously, neovascularization and osteoclastogenesis occur in the same region. The transition is synchronized, at least in part, by soluble morphogens expressed by chondrocytes (163). Osteoclasts degrade the cartilaginous matrix to make way for further bone development, and osteoblasts synthesize a bone-specific matrix, using the degraded cartilaginous matrix as a scaffold.

The coordinated development of osteoblasts and osteoclasts involves cell-cell contact between precursors of both lineages and soluble cytokines and is interconnected with apoptosis of chondrocytes. The entire process is elegantly orchestrated by genetic programs that regulate expression of numerous specific molecules in an ordered spatiotemporal manner (164, 165, 166). Among the myriad morphogens, cytokines, and signaling factors involved in bone formation (155, 156, 157, 158, 164, 165, 166, 167, 168, 169) (Table 1), two pivotal upstream molecules are BMP-2 and the transcription factor core-binding factor 1 [Cbfa1; also called runt-related transcription factor 2, osteoclast-stimulating factor 2, and acute myeloblastic leukemia 3].

BMP signaling utilizes a heteromeric complex of type I and type II transmembrane serine/threonine kinase receptors. Binding of TGF-ß superfamily ligands (TGF-ß, activin, and BMPs) to the type II receptor causes recruitment of the type I receptor into a heterotetrameric structure. There are seven distinct type I receptors identified in vertebrates that can interact with one of five different type II receptors, presenting many combinatorial possibilities for assembly of the receptor complex (173, 177, 178, 179, 180). Because BMP signaling results in diverse biological effects, one might imagine that regulation of downstream gene targets would be a function of the specific heteromeric type I and type II receptor complexes formed. Surprisingly, biological output appears to be exclusively determined by the type I receptor (174, 181). Furthermore, the type I receptor funnels the BMP signal into a single cytoplasmic pathway targeting specific nuclear genes. A series of transphosphorylations allow transmission of the signal to cytoplasmic proteins called Smads (mammalian homologs of mothers against dpp), which then can carry the signal to nuclear effectors. Once nuclear translocation has occurred, highly complex and poorly understood interactions occur between Smad signal transducers and genetic components that result in stimulation or repression of specific target genes to produce biological responses that are variable. Smad heteromeric complexes bind directly to DNA response elements, but high-affinity binding requires cooperative interactions with coactivators and repressors that modulate DNA binding affinities (174, 180, 181, 182, 183, 184). Thus, the BMP signaling pathway resembles an hourglass, with the strategic bottleneck localized at the type I cell surface receptor, and is subject to tight regulation at both the extracellular (185) and intracellular (181, 186, 187, 188, 189, 190, 191, 192, 193, 194) levels. The complexities and dynamic interrelationships of BMP signaling with other signaling pathways have become increasingly apparent (174, 179, 180, 181, 184, 187, 188, 189, 190, 191, 192, 193). Studies examining how BMP signaling participates in arterial calcification are currently in progress.

Cbfa1 is one of the transcription factors that regulates osteoblastic differentiation and bone formation (195, 196, 197, 198, 199). It is clear that Cbfa1 is essential for osteoblast and perhaps also chondrocyte development. Cbfa1 is expressed early in development in cells that have the potential to differentiate into either osteoblasts or chondrocytes and precedes the appearance of bone. Mice with targeted deletion of the gene encoding Cbfa1 manifest a complete lack of osteoblasts and die soon after birth (195, 196). BMPs (including BMP-2) induce the expression of Cbfa1 mRNA (195, 196, 199, 200, 201, 202). BMP signaling and Cbfa1 interact in complex ways that are not fully understood but may be either dependent or independent of interactions with Smads (203, 204, 205, 206).

Although Cbfa1 has been characterized as the "master" upstream osteoblast transcription factor, recent evidence indicates that other mechanisms critically regulate osteoblast formation and function. For example, mice deficient in the low-density lipoprotein (LDL) receptor-related protein 5 demonstrate an osteopenic phenotype due to decreased numbers and activity of osteoblasts (207). Another member of the runt-related transcription factor family, CBFß (core binding factor ß; also called polyoma enhancer binding protein 2ß) was recently shown to have a critical role in bone development (208, 209). The CBFß–/– mutation is embryonic lethal in mice, due to hemorrhage and lack of hematopoiesis (210, 211). CBFß is required for myeloid and lymphoid differentiation but does not play a critical role in erythroid differentiation (212). CBFß is expressed in developing bone and interacts closely with Cbfa1. Knock-in mice partially expressing CBFß demonstrate a phenotype similar to that of Cbfa1–/– mice, i.e., delayed chondrocyte differentiation and endochondral and intramembraneous ossification, but the phenotype is less severe than that exhibited by Cbfa1–/– mice.

Pluripotent mesenchymal tissues are capable of differentiation into adipocytes, myoblasts, chondrocytes, or osteoblasts. Lineage commitment depends upon coordinated expression of sets of specific transcription factors and concomitant suppression of other genetic programs that specify alternative cellular fates. PPAR and C/EBP genes are required for adipogenesis (213, 214), MyoD and myogenin specify myoblast cell fate (215), and Sox5, Sox6, and particularly Sox9 genes are essential for development and maturation of chondrocytes (160, 216).

BMPs are powerful inducers of ectopic bone formation when injected sc at nonbone sites (172, 217). The mechanism involves transformation of mesenchymal cells into osteoblasts in a manner reminiscent of endochondral bone formation. These observations are physiologically relevant. In developing organisms, BMP signaling regulates early commitment and differentiation of pluripotent mesenchymal progenitors by induction of complex genetic programs that are not yet completely understood (159). Among the many gene targets of BMP signaling identified to date, three transcription factors—Distal-less5 (Dlx5), Cbfa1, and osterix (Osx)—are now considered to be the master genes essential for differentiation of mesenchymal progenitors into terminally differentiated osteoblasts. Determining the precise function and regulation of these genes in the adult organism has been limited by the fact that targeted deletion of these genes is either embryonic lethal (Dlx5) or produces mice that are not viable and die shortly after birth (Cbfa1 and Osx). In mice deficient in Cbfa1 (195) or Osx (218), mesenchymal cells are unable to differentiate into functional osteoblasts and do not deposit bone matrix. Cbfa1 and Osx (219) expression are both induced by BMPs. Cbfa1 expression in Osx–/– mice is normal (218), suggesting that Cbfa1 lies upstream of Osx. Recent data indicate that Dlx5 is a direct gene target of BMP-2 signaling and that Dlx5 is a key upstream regulator of both Cbfa1 and Osx (220). One proposed model suggests that Dlx5 induces first Cbfa1 and then Osx in a temporally controlled manner that is not yet understood. The primary role of Cbfa1 appears to be to provide a pool of osteoblast progenitors, and Osx in turn is largely responsible for terminal differentiation of these preosteoblasts into fully functional osteoblasts. However, Cbfa1 also participates in functional activities of osteoblasts (155, 196, 221). In addition, mice overexpressing Cbfa1 manifest osteopenia resulting from increased osteoblast formation but also increased osteoclastogenesis by a mechanism that appears to involve enhanced expression of receptor activator of nuclear factor B (RANK) ligand (RANKL), macrophage colony-stimulating factor (M-CSF), and matrix metalloproteinase-13 (MMP-13) (collagenase 3) and decreased synthesis of osteoprotegerin (OPG) by osteoblasts (222).

Muscle segment homeobox 2 (Msx2) is another homeodomain transcription factor that appears to be an important downstream target of BMP signaling modulating osteoblast development from mesenchymal pluripotent progenitors (223). In vitro, Msx2 was strongly up-regulated by BMP-2, and it was demonstrated that Msx2 increased primary aortic myofibroblast expression of Osx 10-fold compared with control conditions. Cbfa1 expression was not changed by Msx2, but alkaline phosphatase was increased 50-fold, whereas genes associated with adipogenesis were suppressed (223). These results are most consistent with the interpretation that Dlx5 and Msx2 are two important gene targets of BMP signaling in osteoblast development, but that Dlx5 acts earlier than Msx in differentiation and perhaps function of osteoblasts. Cbfa1 (224, 225) and Sox9 (225) are expressed in arterial tissues, and increased expression of Cbfa1 is associated with calcification of atherosclerotic plaque (224, 225, 226, 227). However, expression of Osx, Dlx5, and Msx2 in arterial tissues has been reported.

Bone is constantly being degraded throughout life as a part of normal remodeling and to maintain mineral and electrolyte concentrations within physiologically acceptable limits. Bone formation is normally closely linked to bone resorption; however, there is net bone formation during the first two decades of life. Total bone mass peaks around the third decade, and thereafter bone degradation exceeds formation and there is a gradual loss of bone mass for the remainder of the life span. Differentiation and maturation of osteoclasts are critically regulated by multiple secreted molecules and transcription factors, including c-fos, nuclear factor-B, PU.1 (also called Spi-1; the protein encoded by the purine-rich box-1 gene), and microphtalmia transcription factor, and involve cell-cell interactions with osteoblasts or bone marrow stromal cells (155, 156, 157, 158, 228, 229). Mature osteoclasts stain positively for tartrate-resistant acid phosphatase (TRAP) and express a number of characteristic molecules, including calcitonin receptor (CTR), cathepsin K (cat K), carbonic anhydrase II (CA-II), the proton pump ATP6i, and the vß3 integrin. Osteoclasts are traditionally considered to be present only in bone; however, some osteoclast progenitors circulate in the mononuclear fraction (230, 231, 232, 233, 234) and are recruited by specific interactions with vascular endothelial cells (ECs) (235). A number of genetically altered mouse models with bone disorders characterized by altered bone resorption or osteoblast activity have been developed and widely used to study bone diseases such as osteoporosis and osteopetrosis (236).

B. The active model of arterial calcification
Pathologists have long known that heterotopic calcification such as that seen in atherosclerotic arteries can evolve into mature bone tissue histomorphologically indistinguishable from skeletal bone (25, 144, 145, 237, 238) (Fig. 5). The active model of arterial calcification evolved partly from these observations, stimulated in large part by studies of Bostrom et al. (170), who reported the existence of pluripotent arterial cells that they named calcifying vascular cells (CVCs), which were immunologically distinct from other arterial cells. CVCs colocalized with expression of bonerelated proteins including BMP-2, a potent osteogenic agent, and also could form mineralized nodules in cell culture under certain conditions (170, 239, 240). Subsequently, several groups independently confirmed these in vitro results but also demonstrated that, besides CVCs, arterial SMCs could also form mineralized nodules and express matrix proteins typically found in bone (226, 241, 242, 243, 244, 245). Moreover, expression of bone matrix-associated proteins appears to colocalize with a number of cell types in arterial tissue sections (224, 225, 227, 246, 247, 248, 249). For these reasons, the precise identification of the cell type(s) participating in arterial calcification in vivo remains uncertain.

Recently, Engelse et al. (227) found that expression of Cbfa1 was largely restricted to monocyte-macrophages and, to a lesser extent, SMCs in human atherosclerotic lesions. It is unlikely that cells derived from the mononuclear phagocytic lineage give rise to osteoblast-like cells within the artery. However, this same group recently reported that in bone, osteoclasts (which are derived from mononuclear phagocytic precursors) demonstrate immunopositivity for Cbfa1 in the absence of any detectable Cbfa1 gene expression (i.e., mRNA transcripts) (250). What at first appeared to be Cbfa1 protein expression by osteoclasts was actually the result of phagocytosis and incomplete digestion of cells derived from the osteogenic/chondrogenic lineages, which are well known to express Cbfa1. It is possible that a similar process accounts for apparent Cbfa1 protein expression in arterial monocyte-macrophages. However, because Cbfa1 transcripts were detected in the MM6 monocytic cell line and in a subset of macrophages in a limited number of human arterial sections (227) but not in bone osteoclasts (250), more definitive resolution of this issue will require further study. Cbfa1 is also expressed in both the intima and media in the arteries of patients with ESRD and both medial and intimal (atherosclerotic) calcification (224). Numerous other reports have consistently confirmed that in atherosclerotic arteries, and particularly areas of plaque where calcification occurs, increased expression of proteins normally restricted to developing bone is observed (142, 225, 246, 247, 248, 249). Nevertheless, the validity of the active model has not been tested in vivo, and alternative models, as described below, have been proposed.

Studies of both human arteries and animal models of atherosclerosis have revealed the existence of cells that are phenotypically similar to all major cell types involved in endochondral bone formation—chondrocytes, osteoblasts, and osteoclasts (144, 145, 237, 238, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259) (Figs. 4–7). Furthermore, these cells possess the required signaling pathways and express proteins normally associated with their analogs in bone (140, 141). Mineralization occurs in bone via matrix vesicles, and these have also been described in arterial tissues (260, 261, 262, 263) (Fig. 7). The mineral composition of bone (hydroxyapatite) is chemically very similar to that observed in calcific deposits in atherosclerotic arteries (170, 264, 265, 266, 267). Collectively, these findings strongly support a model wherein arterial calcification occurs in plaque microenvironments in a manner that recapitulates osteogenesis.

View larger version (112K): [in this window] [in a new window]   FIG. 7. Arterial calcification in animal models. Some animals may spontaneously develop arterial calcification with time. A, A maximum intensity projection from a micro-CT scan of an aged (26 months) rat heart. Calcification is evident in all major epicardial coronary arteries. B, A cross-section of one of the coronary arteries with von Kossa staining. Calcification appears as dark regions in the arterial wall. Sections were not decalcified before methylmethacrylate embedding and Goldner’s-Masson-Trichrome (C and D, blue-green color) or von Kossa (E, black color) staining. Red regions are unmineralized matrix. Many animal models also manifest evidence of cartilage-like tissues and chondrocytes. For example, panels C–E show cartilage (arrow in C; magnified in D and E) within the vascular wall from an aged oophorectomized female rat. A diffuse mineralization pattern can be seen (C and D), with some punctate areas as well. On the other hand, some animal models exhibit calcification largely or exclusively confined to the media, typically in association with elastic fibers. Sections of abdominal aorta from rats treated with warfarin and vitamin K for 2, 3, 4, or 5 wk (F–I, respectively) and stained with von Kossa (black) shows progressive mineralization involving the media, with little or no neointima present. J and K, Aortic sections from rats treated with vitamin D alone for 96 h (J) or vitamin D plus the osteoclast inhibitor SB 242784 (K). Both sections are stained with hematoxylin and eosin, as well as with von Kossa. L, Aortic sections from rats treated for 8 wk with warfarin and vitamin K1 and stained with von Kossa. Mineralization of elastic fibers is evident, which eventually develops into larger calcium deposits, but involves only the medial arterial layer because these animals have no intimal atherosclerotic plaque. J–L, The arterial lumen. Medial calcification can, in some animal models, appear similar to that typically seen in intimal tissues. M, Hematoxylin and eosin staining of aortic section from OPG–/– mice shows calcified lesions (dark purple) in the media and also involving intimal tissue. N–R, Large, multinucleated cells can be seen in these lesions (yellow arrows) that stain positive for cat K (P) and TRAP (Q). However, other multinucleated cells in these lesions are negative for cat K, TRAP, and the macrophage marker F4/80 (R), suggesting dedifferentiated cells or pluripotent cells of undetermined origin and fate. MGP–/– mice exhibit diverse features of arterial calcification, but again this is almost completely limited to the media because little or no intima is present. S–W, Aortic sections from MGP–/– mice. Mineralization of elastic lamellae in the aorta is evident (S) (von Kossa staining for mineral appears black). T, Mineralization of internal elastic lamina (arrow) in a coronary artery. Note that calcification is not intimal but rather in the medial layer. U, A transmission electron micrograph (TEM) of aortae from MGP–/– mice showing extensive calcification of elastic fibers in the media (cEF, calcified elastic fiber; nEF, noncalcified elastic fiber). Note the calcification "front" that appears to be advancing along the length of the fiber (arrowhead). V, A TEM of MGP–/– aorta shows chondrocytes (ch) surrounded by hyaline cartilage and type II collagen fibrils (arrows), proteoglycans (arrowheads), and matrix vesicles (inset). W, Aortic cartilaginous metaplasia in an MGP–/– mouse with chondrocytes (arrows) in the midst of metachromatic cartilage matrix. Augmentation of aortic calcification is observed in MGP–/– OPN–/– mice when compared with MGP–/– mice. X, An aortic section from an MGP–/– OPN+/+ mouse stained with von Kossa. Y, A similar section from a double knockout (MGP–/– OPN–/–) mouse. Z, Fragmentation of the elastic lamellae (arrows) can be seen in aortae obtained from MGP–/– OPN–/– double knockout mice (panels X–Z: M, media; Ad, adventitia; L, lumen; asterisks, calcium deposition). AA and BB, TEM shows mineralization of intracellular (AA, arrowheads) or extracellular (BB, arrowheads) membranous compartments adjacent to sc implanted glutaraldehyde-fixed aortic valve leaflets in OPN–/– mice. Mineralization was also observed in extracellular matrix vesicles (BB, arrows). Macrophages (blue stain) and multinucleated giant cells localized to the region and stained positive for the osteoclast-related enzyme CA-II (CC, brown stain; inset shows a higher magnification of the boxed region). DD, Both multinucleated CA-II-positive cells and mononuclear phagocytes adjacent to implants. These colocalized with regions of developing mineral deposits stained with Alizarin red in OPN+/+ mice (EE). FF, A higher magnification of the boxed region. GG, There was also colocalization of OPN staining (brown) with mineral deposits (Alizarin red stain) in OPN+/+ mice. [Panels A–E reproduced with permission from L. A. Fitzpatrick et al.: Endocrinology 144:2214–2219, 2003 (253 ); panels F–I reproduced with permission from P. A. Price et al.: Arterioscler Thromb Vasc Biol 18:1400–1407, 1998 (70 ); panels J and K reproduced with permission from P. A. Price et al.: Circ Res 91:547–552, 2002 (51 ); panel L reproduced with permission from R. Essalihi et al.: Am J Hypertens 16:103–110, 2003 (72 ); panels M–R reproduced with permission from H. Min et al.: J Exp Med 192:463–474, 2000 (341 ); panels S–W adapted with permission from G. Luo et al.: Nature 386:78–81 (127 ); panels X–Z reproduced with permission from M. Y. Speer et al.: J Exp Med 196:1047–1055, 2002 (134 ); panels AA–GG reproduced with permission from S. A. Steitz et al.: Am J Pathol 161:2035–2046, 2002 (248 ).]

M-CSF is a key cytokine involved in survival of osteoclast progenitors and differentiation toward a mature functional osteoclast phenotype (157, 158, 229, 270). Earlier studies with mice lacking both M-CSF and apolipoprotein (apo) E suggested a potential role for M-CSF in arterial calcification (271). Apo E-deficient mice may develop atherosclerotic lesions that histologically exhibit many features of human lesions (272, 273), including calcification (254, 255, 271, 274). Mice harboring a naturally occurring structural mutation in the M-CSF gene exhibit osteopetrosis as a result of an absence of osteoclasts (275, 276). A striking finding in our study was that approximately one third of double knockout mice lacking both M-CSF and apo E developed massive arterial calcification but no atherosclerotic lesions in the vessel wall (271). This suggested the possibility that the development of calcification in atherosclerotic lesions may, in part, be the result of decreased osteoclastic activity due to lack of M-CSF. Accumulating evidence reviewed later in this section now increasingly supports this intriguing possibility.

M-CSF and RANKL signaling are both necessary and sufficient for osteoclastogenesis and function. M-CSF is a multifunctional protein that regulates the differentiation, proliferation, survival, and function of mononuclear phagocytic cells (MPCs) in vitro and in vivo by binding to cellular-feline McDonough sarcoma protooncogene (c-fms) (234, 277, 278, 279, 280). All cells of the atherosclerotic vessel wall, including ECs, SMCs, and MPCs, can express M-CSF. M-CSF stimulates the proliferation and survival of macrophages in culture and in vivo, suggesting that induction of M-CSF in atherosclerotic lesions may directly stimulate MPC proliferation. We hypothesize that M-CSF produced locally within developing atherosclerotic lesions is critical to the differentiation of a subpopulation of MPCs toward an osteoclast-like phenotype. This effect of M-CSF on MPCs may take place in an autocrine, paracrine, or juxtacrine manner. Although both M-CSF and RANKL signaling are necessary for osteoclast development, lack of M-CSF alone is sufficient to cause greatly diminished numbers of osteoclasts and osteopetrosis (234, 276, 278, 279).

M-CSF is required not only for survival of MPC precursors, but also for maturation and/or function of osteoclast precursors. This conclusion is supported by studies demonstrating that transgenic overexpression of the antiapoptotic factor B cell chronic leukemia-2 (bcl-2) in M-CSF-deficient mice results in greater than normal levels of circulating monocytes and partial rescue of the osteopetrotic phenotype (281, 282). Stanley and colleagues (283) recently reported that expression of a transgene encoding the full-length M-CSF precursor reconstitutes circulating and tissue levels of the secreted isoform of M-CSF and some of the cell-surface isoform in M-CSF–/– (op/op) mice. Expression of this transgene corrects most of the defects in op/op mice. Furthermore, preliminary studies suggest that compound mutant atherosclerosis-prone mice that are deficient in both M-CSF and apo E and also overexpress bcl-2 (i.e., apo E–/–; M-CSF–/–; bcl-2Tg) develop little or no atheroma, despite marked hypercholesterolemia and normal or greater than normal levels of circulating monocytes (282). These data highlight the central importance of M-CSF in atherogenesis, show that the antiatherogenic effects of M-CSF deficiency are not due to lack of circulating monocytes, and suggest that lack of M-CSF might contribute to arterial calcification by inhibiting development of arterial OLCs, thereby allowing unopposed activity by CVCs (140). Thus, M-CSF may have dual effects: promoting or allowing atherogenesis to develop, and inhibiting calcification.

Absence of RANK also results in osteopetrosis, but rescue of the wild-type phenotype can be achieved by adoptive bone marrow transplantation using recombination activating gene 1–/– bone marrow donors (284). Normal osteoclast precursors are found in rag1–/– mice, suggesting that RANKL signaling is predominantly required for early differentiation of precursors into mature osteoclasts. Because hematopoietic precursors from RANK–/– mice cannot be induced to form osteoclasts in the presence of RANKL and M-CSF, it appears that M-CSF cannot by itself stimulate differentiation and maturation of osteoclast precursors. Retroviral delivery of RANK cDNA into RANK-deficient mice restores normal osteoclast differentiation and function, providing an unequivocal demonstration that osteoclastogenesis is dependent on RANK-mediated signaling. However, the effect of RANK deficiency on arteries has not been fully investigated thus far, and because OPG deficiency results in osteoporosis and paradoxical arterial calcification, it is conceivable that signaling mechanisms regulating OLC maturation and function might be regulated differently in the arterial wall.

The downstream signaling cascade of RANKL/RANK is complex and not fully understood (285, 286, 287, 288, 289). There are at least six receptor-associated adaptor molecules called TNF receptor-associated factors (TRAFs). TRAF6 appears to be crucial to RANKL/RANK signaling (290, 291, 292), because mice deficient in TRAF6 exhibit severe osteopetrosis (293, 294). Downstream of TRAF6, there are at least three divergent signaling pathways; these include the c-jun N-terminal kinase/c-fos/c-jun pathway and the nuclear factor-B pathway (290, 291, 292), both of which are required for osteoclast formation and activation. A third pathway uses cellular-Rous sarcoma virus protooncogene to activate the serine-threonine kinase Akt/PKB; this pathway appears to be primarily directed toward antiapoptotic functions and cytoskeletal reorganization (295, 296).

A number of reports have documented the presence of multinucleated, TRAP-positive cells in arteries (146, 225, 237, 248, 252) (Figs. 5 and 7). Morphological evidence for OLCs is supported by numerous additional lines of evidence (described in the next paragraph). Collectively, these studies suggest that there are OLCs in arteries, but the origin of these cells is unknown. It is possible that OLCs could differentiate from "resident" pluripotent arterial cells, but it is equally likely that they originate from hematopoietic precursors, such as stem cells or cells that have differentiated from mononuclear phagocytic lineage precursors. It is not known what signaling pathways and genetic programs might regulate the development and function of OLCs.

The hypothesis that OLCs exist in the arterial wall and may play a role in plaque calcification is predicated upon multiple lines of evidence, including the following:

1) MPCs are abundantly found in plaque during all stages of atherogenesis (6, 7, 8, 9, 14, 15, 16, 19, 297, 298, 299, 300).

2) MPCs and osteoclasts are both hematopoietic in origin and are closely related to one another (157, 158, 229, 301, 302, 303, 304, 305).

3) Osteoclast progenitors circulate in the mononuclear fraction (230, 231, 232, 233, 234).

4) MPCs (6, 7, 8, 9, 19, 297, 306, 307, 308) and osteoclast precursors (235, 309, 310) are both recruited by ECs from circulating blood.

5) Circulating monocytes (311, 312) and extraskeletal fibroblasts (313) can be induced to differentiate into TRAP-positive multinucleated OLCs that express RANKL and M-CSF mRNA, the osteoclast-associated CTR, and resorb bone (314).

6) Macrophages and multinucleated cells derived from MPCs can degrade mineral deposits under some circumstances (225, 248, 304, 315).

7) M-CSF is expressed in plaque (316, 317) and is essential for mouse and human osteoclast formation (157, 158, 228, 229, 234, 301), and mice deficient in M-CSF develop extensive arterial calcification (271).

8) Circulating osteoclast precursors, MPCs, B cells, and T cells express RANK (232, 295, 318, 319, 320, 321, 322, 323) and c-fms (324, 325, 326), the cell-surface receptor for M-CSF (277, 278, 327).

9) c-fms Expression is, in turn, induced by the transcription factor PU.1 (328, 329), which is essential for osteoclast development (330, 331).

10) RANK, RANKL, and OPG, the decoy receptor for RANKL, are expressed in blood vessels (332, 333, 334, 335, 336).

11) ECs express RANK, RANKL, and OPG (333, 337, 338).

12) OPG and RANKL transcripts are expressed by osteoblastic stromal cells, whereas RANK mRNA is produced by osteoclast precursors (339, 340). All are found in calcified arterial lesions of OPG-deficient mice coinciding with multinucleated TRAP- and cat K-positive cells (336, 341).

13) Osteoclast function is dependent on CA-II (157, 158, 228, 229, 301, 342, 343), and CA-II–/– mice develop osteopetrosis (344) and arterial calcification (345).

14) OPN inhibits hydroxyapatite mineral growth (244, 346, 347, 348) by binding large amounts of Ca²⁺ (50 mol calcium/mol OPN; Ref.349), and is also important in osteoclast differentiation (350, 351, 352) and function (352, 353, 354, 355, 356, 357, 358, 359). Steitz et al. (248) recently reported that OPN–/– mice demonstrate 4- to 5-fold greater calcification around sc implanted aortic valve leaflets compared with wild-type mice. OPN+/+ mice were able to recruit MPCs and multinucleated giant cells around the calcifying implants. These cells expressed CA-II and promoted acidification within calcifying microenvironments and caused a time-dependent reduction in calcification. However, OPN–/– mice were unable to express significant levels of CA-II, could not acidify calcifying areas of implanted valve leaflets, and demonstrated significantly greater quantities of calcification around implanted valve leaflets. Matsui et al. (256) recently provided corroborative support, reporting that male mice with homozygous genetic deficiency in both OPN and apo E exhibited greater calcification than either OPN+/–; apo E–/– mice or apo E–/–, although there were no significant differences in atherosclerotic plaque area among the three groups.

15) TRAP- and cat K-positive multinucleated OLCs have been observed in mineralized arteries of apo E–/– (360) and OPG–/– (336, 341) mice and in human aortic (225) and carotid artery (225, 237, 238) plaques.

Collectively, these data suggest the presence and possible participation of OLCs in arterial calcification and provide the rationale for a detailed investigation of mechanisms underlying the action of putative OLCs (140) in the diseased vessel wall.

However, several other studies argue against the OLC hypothesis. The OLC hypothesis would predict that experimental manipulations that enhance osteoclast function would result in decreased calcification and inhibition of osteoclast function would be associated with increased calcification. However, OPG–/– mice develop arterial calcification (336, 341) (Fig. 7). Observational expression analyses indicate that as atherosclerosis progresses and regions of calcification develop, RANKL expression increases. The OLC model would predict that this should enhance OLC development and thereby inhibit calcification. However, cell culture studies show that stimulation with RANKL can increase Cbfa1 binding to DNA and could thereby induce expression of osteoblast-related genes (361). These findings have led to the proposal that RANKL stimulates calcification, but OPG tends to inhibit RANKL actions in promoting mineralization (361). Other data are also not readily explained by the OLC model. For example, pharmacological inhibition of osteoclast function by three independent mechanisms results in less arterial calcification in the warfarin/vitamin K rat model of arterial calcification. These include inhibition of the V-H⁺-ATPase (51) (vacuolar proton ATPase; an enzyme required for osteoclasts to acidify substrate before resorption; Refs.362, 363, 364), treatment with bisphosphonate osteoclast inhibitors alendronate and ibandronate (52, 69), and treatment with OPG (68). However, several caveats should be noted. First, the dose of drug required to inhibit V-H⁺-ATPase (51) was four times higher than the dose that inhibits experimental osteoporosis in the same rat model (365). Second, both OPG–/– mice and rats treated with warfarin and vitamin K exhibit medial (not intimal) calcification similar to Mönckeberg’s sclerosis. The relevance of these results to intimal calcification in the setting of atherosclerosis is therefore uncertain. Third, if inhibition of osteoclasts increases both medial and intimal plaque calcification, then it would be anticipated that patients being treated for osteoporosis with bisphosphonates would have greater amounts of arterial calcification. Similarly, warfarin treatment alone would, as in the rat model, be expected to increase arterial plaque calcification in patients on long-term anticoagulant therapy. No clinical studies demonstrating this to be the case have been reported. Hill et al. (366) recently reported that coronary calcification was not accelerated in patients taking alendronate; but the study included small numbers of patients (56 patients and 56 matched controls), the study duration (24 months) may have been insufficient to detect significant changes in coronary calcification, and many patients had low baseline calcium scores, which would tend to adversely affect retest reproducibility, a well-known limitation of computed tomography (CT) coronary calcium scanning studies (367).

Larger amounts of atherosclerotic coronary calcification are associated with increased risk of myocardial infarction and sudden coronary death (21, 22, 24). It might therefore be anticipated that, if patients taking bisphosphonates or warfarin are more prone to accelerated calcification, this might be reflected in greater risk of such coronary events. The effects of bisphosphonates on coronary risk have not been reported. However, several reports indicate that patients taking warfarin are at decreased risk of cardiac events (368, 369, 370), but the effect of confounding variables—such as the effects of antithrombotic therapy itself on coronary risk—might more than counteract any increased risk secondary to accelerated arterial calcification. One approach to better understanding these issues would be to assess coronary artery calcification progression in patients receiving these agents over time, because medial calcification has not been reported to occur in the coronary arteries. Changes in coronary calcification in patients on bisphosphonate and/or warfarin therapy could then be compared with suitable controls after adjusting for coronary risk factors and other variables that could affect progression of calcification. Such studies have not yet been performed. Obviously, it will be necessary to test these hypotheses directly in clinical studies as well as in animal models of atherosclerotic plaque calcification.

Studies with genetically altered mice strongly suggest a role for RANKL signaling in arterial calcification, but the possible mechanisms are both unclear and untested. RANK, RANKL, and OPG are expressed in arteries (146, 332, 336, 341). Mice deficient in OPG develop not only osteoporosis but also arterial calcification (336, 341). Osteoporosis, but not arterial calcification, can be rescued by injections of OPG in OPG–/– mice, but both osteoporosis and arterial calcification were prevented by overexpression of the OPG transgene (341). With all other factors constant, if RANKL signaling promotes arterial OLC development and function, then depletion of the inhibitory decoy OPG should produce the opposite effect, namely enhanced mineral resorption. There are several possible explanations. For example, RANKL and RANK are not detected in normal mouse arteries, but OPG is expressed (341). However, because RANKL, RANK, and OPG transcripts are expressed in calcified plaque, the calcification process may up-regulate RANK and RANKL expression and activate RANKL signaling. The inhibitory effect of OPG could suppress the ability of RANKL signaling to stimulate differentiation and function of OLCs; alternatively, induction of RANKL signaling may be insufficient to counter the mineral deposition occurring in plaque. An unlikely possibility is that RANKL signaling could promote mineral deposition in arteries. Alternatively, in addition to competitive inhibition of RANKL signaling, OPG might have independent functions that promote OLC maturation, survival, and/or function.

In summary, a number of lines of indirect evidence are consistent with the conclusion that OLCs participate in intimal plaque calcification, whether by actively inhibiting calcification or degrading existing mineral deposits (140). However, this hypothesis also has not yet been directly tested. Moreover, even if OLCs inhibit or resorb arterial mineral deposits, this does not exclude the possibility that calcification is also regulated by other mechanisms.

D. The passive physicochemical model of arterial calcification
It is clear that calcium and phosphate ions in biological fluids are in a metastable state in concentrations that are near the point where precipitation of mineral salts occurs (i.e., the K_sp or, more practically, the ion product). To prevent the spontaneous formation of Ca²⁺:PO₄^–3 solid phases in extracellular fluids, it is thought that a number of proteins inhibit precipitation or chelate/sequester ions to lessen their bioavailability. There are many such inhibitors, and prominent among these are MGP in the extracellular matrix and albumin and ₂-Heremans-Schmid glycoproteins (AHSGs)/fetuins in serum. The final process of mineral deposition in bone involves a mechanism whereby normal inhibitory mechanisms are blocked in a carefully regulated manner both temporally and spatially (371, 372, 373, 374, 375, 376).

Based on these concepts, the passive physicochemical model was first proposed by Vermeer and colleagues (149, 377, 378, 379, 380, 381, 382) and postulates that normally there are inhibitors present in arteries that prevent precipitation of calcium mineral salts, at least partly by chelating calcium cations. Several proteins known to be expressed in arteries modulate precipitation of calcium salts in the extracellular fluid, notably proteins that contain unusual glutamine residues with an additional carboxy group added in an enzymatic reaction catalyzed by -carboxylase (383, 384). Glutamine residues carboxylated at the position are called Gla residues. Proteins containing Gla residues are known as Gla proteins, and Gla residues are an important functional feature of both hemostatic clotting factors and bone proteins such as MGP and BGP (or osteocalcin) (385, 386, 387, 388, 389). -Carboxylase is widely distributed in tissues, including arteries, and catalytic activity is less in atherosclerotic arteries compared with either normal arteries or liver (149). Recently, it was reported that the relative availability of two distinct -carboxylase cofactors may help explain why calcification tends to occur in arteries and much less frequently in other tissues (390). At least two forms of vitamin K can participate in the reaction that produces Gla residues: phylloquinone (K1) and menaquinone (MK)-1. In vitro, both are roughly equivalent in cofactor activity. But in the rat warfarin/vitamin K arterial calcification model, Spronk et al. (390) showed that MK-4 inhibits warfarin-induced arterial calcification, but K1 does not. These investigators concluded that differences in both tissue distributions and utilization efficiencies of K1 and MK-4 may contribute to the effects observed in the rat warfarin/vitamin K model and potentially also to forms of calcification seen in other experimental models and in human arteries.

The passive physicochemical model has received support from several independent sources. First, both expression and activity of -carboxylase in atherosclerotic arteries is inhibited, and this would be expected to produce undercarboxylated Gla proteins (149, 378, 381, 382). Second, in mice with targeted deletion of the gene encoding MGP, massive arterial calcification is seen, and the mice die by the age of about 60 d from aortic rupture and hemorrhage (127, 128). Third, undercarboxylation of MGP produces arterial calcification in a rat model (68, 69, 70, 71, 72). The activity of -carboxylase can be decreased by administering warfarin and vitamin K. Warfarin is a potent anticoagulant that inhibits the -carboxylation of Gla proteins, and because several clotting factors are Gla proteins and their function depends upon -carboxylation of Glu to Gla residues, undercarboxylation of Gla-containing clotting factors results in the maintenance of an anticoagulated state. There are, however, several Gla-containing proteins involved in bone formation as well, and their function in bone depends on the carboxylation status of the Glu residues. When warfarin is combined with vitamin K, the result is that undercarboxylation of MGP occurs without significant effects on clotting factor Gla proteins (68, 69, 70, 71, 72). Rats treated with warfarin and vitamin K demonstrated impressive arterial calcification (Fig. 7). Together, these findings would appear most consistent with a homeostatic role for MGP wherein MGP prevents precipitation of calcium in arteries. Calcification would occur only when MGP and possibly other calcium chelators and inhibitors are unable to prevent ionic concentrations in the extracellular fluid from reaching the K_sp or the ion product for precipitation.

There are several dilemmas with this model, however. First, calcification in atherosclerotic arteries occurs in the intima (11, 25, 141) (Figs. 3 and 5). However, in the MGP knockout mouse (127, 128, 134) and in rats treated with vitamin K and warfarin (68, 69, 70, 71, 72), calcification is largely confined to the media because these animals are naturally resistant to atherosclerosis and have little or no intimal layer in their arteries (Fig. 7). Histologically, the pattern of calcification appears less like that observed in plaque (Fig. 5), but rather resembles that seen in Mönckeberg’s calcinosis (32, 33, 34, 35, 36, 39, 40, 41, 42, 43, 44, 45) (Fig. 5). Furthermore, Keutel syndrome is a human inherited disorder that results in a nonfunctional MGP gene (391). However, this syndrome is not associated with the massive arterial calcification that is observed in the Mgp knockout mouse (127, 128, 134). In vitro, mineralization of vascular cells is accompanied by high expression levels of MGP (245), a finding that is difficult to reconcile with a model wherein MGP inhibits mineralization. On the other hand, cell culture results may be misleading, but collectively, these findings raise significant doubt as to the validity and relevance of the passive physicochemical model.

Nevertheless, the passive model may overlap with other models and might ultimately be proven valid. For example, in MGP–/– mice, there are clear indications that the mechanism of arterial calcification cannot be easily reduced to simple apatite crystallization arising from ionic precipitation of Ca²⁺ and PO₄^–3 ions when MGP inhibition is removed. First, chondrocytes, hyaline cartilage, type II collagen, cartilaginous matrix, cartilaginous metaplasia, and matrix vesicles are all found in calcified arteries of MGP–/– mice (127, 128, 134) (Fig. 6). Second, there may be a link between MGP and BMP signaling, and it has been proposed that MGP is an extracellular inhibitor of BMP ligand (392). Recent evidence from cell culture studies (135, 136, 393) and intriguing results from a rat model of arterial calcification (394) are also consistent with this notion. Sweatt et al. (394) reported that the Gla-containing domain of MGP binds recombinant BMP-2 in the presence of Ca²⁺. Immunohistochemical analysis of aged rat aortae showed high concentrations of MGP associated with calcified lesions. Aortic MGP was found to be poorly carboxylated, which decreased its affinity for BMP-2. These results are consistent with the notion that undercarboxylation of MGP may play a role in facilitating arterial calcification secondary to decreased inhibition of BMP-2. A similar mechanism could also contribute to the arterial calcification observed in MGP–/– mice. In future studies, it will be important to more completely determine how atherogenesis affects MGP carboxylation and downstream BMP signaling effects within plaque microenvironments.

Postnatal transgenic expression of OPG prevents arterial calcification (341), and in the warfarin/vitamin K or the vitamin D rat models of arterial calcification, administration of OPG inhibited the development of medial arterial calcification (68). These findings have led to the suggestion that OPG might function in arteries to inhibit calcification, in a manner similar to that proposed for MGP. However, it is not clear how OPG might directly prevent mineralization. MGP contains five Gla residues that are thought to directly chelate calcium ions (395), but OPG contains no structural motif that might chelate or associate with ionic calcium (396, 397). It would therefore appear that if OPG functions similarly to MGP in arteries, then it likely does so in a manner that is mechanistically distinct and may not be direct. However, the possibility that OPG and MGP directly interact to cooperatively or synergistically inhibit mineralization cannot be excluded.

In any case, MGP–/– mice exhibit extensive medial arterial calcification (127, 128, 134) despite, presumably, the presence of OPG (Fig. 7). It would be of considerable interest to determine how expression of OPG is related to the degree of arterial calcification in apo E–/– mice, which spontaneously develop intimal plaque calcification. It is not clear whether the primary function of OPG in arteries is to inhibit biomineralization, and even if this is, in fact, its main function in arteries, OPG may not directly cause physicochemical inhibition. Further studies are needed to evaluate these possibilities and clarify the potential roles of both MGP and OPG in arterial calcification.

Besides Gla proteins such as MPG, there are other calcium-chelating proteins that could potentially play a role in suppressing mineralization. Most calcium-chelating proteins, including MPG, are thought to act predominantly or even exclusively in limited microenvironments. At least one reason for this is that the chemical nature of these proteins makes them poor candidates for playing any kind of general role in tissue homeostasis. For example, MPG is poorly soluble, is synthesized locally, and is predominantly found bound to extracellular matrix elements (398, 399). However, other proteins may well play a systemic, homeostatic role by circulating through tissues and preventing mineral deposition. AHSG is notable in this regard. AHSG is a cysteine protease inhibitor and a member of the cystatin superfamily of protease inhibitors (400, 401). AHSG circulates in serum, is abundantly expressed in both mammalian and nonmammalian species, and accumulates in bone because of a high affinity to hydroxyapatite (402, 403). It has been suggested that AHSG prevents de novo mineralization by transiently (i.e., for several hours) forming "calciprotein" spherical colloidal complexes consisting of AHSG and basic calcium phosphate, the precursor to hydroxyapatite. Notably, however, AHSG does not appear to possess the capacity to dissolve mineral deposits such as basic calcium phosphate once they have formed (403, 404). These features make AHSG an attractive candidate for a general inhibitor of mineral precipitation in serum and tissues and raise the possibility that AHSG might play an important role in arterial calcification in a manner most consistent with the passive physicochemical model. To evaluate the validity of this notion, Jahnen-Dechent et al. (405) generated mice with a targeted genetic deletion of the gene encoding AHSG and found that AHSG–/– mice exhibit a mild phenotype; however, alizarin red staining of bone revealed the existence of soft-tissue calcification in some female mice. In a subsequent report (406), these investigators bred the AHSG-null mice with the DBA/2 strain, which is known to be sensitive to dystrophic mineralization (407), and fed the resulting progeny either standard rodent chow or a vitamin D-enriched diet. After backcrossing onto pure C57BL/6 or DBA/2 strains for at least 10 generations, the AHSG-null mice on the DBA/2 background exhibited reduced breeding capacity and approximately 25% 6-month mortality. This was associated with extensive ectopic calcification of soft tissues, including the kidneys, myocardium, lung, and skin, which did not occur in AHSG+/+ littermates. Calcification appeared particularly prominent in organs such as the kidneys that are involved in the secretion or transport of mineral-rich fluids or in the generation of local pH changes. There were a number of systemic metabolic abnormalities observed in these mice, including high blood pressure, hydronephrosis, marked albuminuria, renal failure, osteopenia, and secondary hyperparathyroidism. Serum electrolyte concentrations were surprisingly normal, a finding that underscores the importance of AHSG in preventing mineral precipitation independently of ion concentrations per se. Diet did not appear to be a major factor in producing the ectopic calcification phenotype, or at least the effects of diet were subtle compared with the influence of the background DBA/2 strain in the context of the AHSG-null mutation. Although these investigators did not examine the arteries, it is likely that arterial calcification more akin to that observed with MGP–/– would be present. Because AHSG-deficient mice against either the C57BL/6 or DBA/2 backgrounds do not develop atherosclerosis, the possible participation of AHSG in plaque calcification has yet to be established.

Other mechanisms have been proposed to account for arterial mineralization as well. Apoptotic bodies and/or matrix vesicles derived from SMCs or macrophages in plaque are thought to be a source of mineral nucleation sites (33, 408) (Figs. 4 and 7). Cells undergoing apoptosis liberate both calcium and phosphate ions and would be anticipated to promote calcification. In support of this notion are data demonstrating that extracellular calcium ion concentrations are markedly increased in inflammatory tissues (409), and cell culture studies have shown that increased phosphate ion concentrations can induce osteogenic differentiation in vascular SMCs (348). Lipids in plaque might participate in calcification by stimulating osteogenic differentiation and/or serving as a nidus for mineralization (410, 411) (Fig. 4). Matrix vesicles have been observed in the medial layer of calcifying arteries in association with elastin (262, 263, 412, 413), and elastic fibers such as those found in the media enhance precipitation (371). A recent report demonstrated that treatment of rabbit iliac arteries with balloon angioplasty and cryotherapy resulted in some cases in the formation of bone and cartilage near the border of the neointima and media (249). These regions colocalized with marked expression of BMP-2 and appeared to be centered in subregions where there was disintegration of the internal elastic lamina and other elastic fibers. However, this model was a simulation of restenosis and not de novo atherosclerosis. Collectively then, these data appear to be more relevant to medial calcification, which is histologically distinct from intimal calcification and often does not coincide with atherosclerotic plaque (see Section III.A).

E. A unified model
It is entirely possible, perhaps even likely, that more than one mechanism of plaque calcification is operative and that these might be altered by the chronic inflammation that is often seen in atherosclerotic plaque (141). During bone formation, there are complex mechanisms orchestrated by sequential activation of genetic programs that carefully regulate when, where, in what spatial direction, and how long mineral precipitation occurs. When a fracture occurs, for example, bone formation at the site is not randomly directed, but healing occurs in such a way that the morphological characteristics of bone are preserved (269, 414, 415). As described above, bone formation is mediated by osteoblasts and directly opposed by osteoclast-mediated bone resorption. Normal bone formation and remodeling involves concomitant activity of both osteoblasts and osteoclasts, and there is considerable coordination between the two such that the processes are synchronized. Furthermore, endochondral bone formation depends upon resorption of matrix during the transition from the cartilaginous intermediate stage to prepare the way for osteoblast precursors to develop and begin to deposit matrix and mineral upon the cartilaginous matrix template. These considerations imply that distinct yet concurrently operative mechanisms may have relevance to arterial calcification as well. It seems likely that in arterial calcification, proposed models are not mutually exclusive.

	IV. Endocrine, Pharmacological, and Lipid Influences on Arterial Calcification

Top Abstract I. Introduction: Clinical... II. Types of Arterial... III. Molecular Mechanisms of... IV. Endocrine, Pharmacological,... V. Genetic Determinants of... VI. Conclusions References

 
A. Parathyroid hormone and vitamin D
PTH and the active form of vitamin D [1,25-dihydroxyvitamin D₃ (1,25-(OH)₂D₃)] are the principal regulators of calcium homeostasis in bone. PTH stimulates the release of calcium and phosphate from bone. In the kidney, PTH stimulates reabsorption of calcium and inhibits the reabsorption of phosphate. PTH also stimulates the action of 1-hydroxylase, which enhances the synthesis of 1,25-(OH)₂D₃ in the kidney. 1,25-(OH)₂D₃ in turn increases the intestinal absorption of calcium and phosphate. Thus, PTH maintains blood calcium and phosphate concentrations via multiple mechanisms.

The actions of PTH on bone are complex and only partially understood. Osteoblasts, bone marrow stromal cells, hematopoietic precursors of osteoclasts, and mature osteoclasts all respond to the actions of PTH. Prolonged administration or increased secretion of PTH (such as in primary hyperparathyroidism) increase osteoclast number and activity and thus promote the release of calcium, phosphate, and matrix components from bone. In contrast, short-term or intermittent administration of PTH increases bone formation through its anabolic actions on the osteoblasts and their precursors. PTH alters osteoclast maturation and function indirectly by stimulating expression of RANKL.

The main action of 1,25-(OH)₂D₃ is to maintain the serum calcium level within the normal range, by increasing efficiency of intestinal calcium resorption and by inducing stem cells in bone to become mature osteoclasts, which then mobilize calcium from bone. Osteoclast function is also indirectly regulated by 1,25-(OH)₂D₃ by its action on osteoblasts. 1,25-(OH)₂D₃ increases the expression of alkaline phosphatase, OPN, and osteocalcin, all of which play important roles in bone mineralization. By a process that is incompletely understood, 1,25-(OH)₂D₃ also promotes the mineralization of osteoid laid down by osteoblasts (416). The actions of 1,25-(OH)₂D₃ thus maintain calcium homeostasis through its actions on osteoblast function and the differentiation of monocytes into osteoclasts. In addition, dependent on the serum calcium level, 1,25-(OH)₂D₃ can be a potent stimulator of either bone resorption or bone formation.

Vitamin D is metabolized by three different cytochrome P450 enzymes. One of these enzymes, cytochrome 24, converts 25-hydroxyvitamin D to 24,25-dihydroxyvitamin D [24,25-(OH)₂D] and other more polar metabolites. Transgenic rats that constitutively express the cytochrome 24 gene exhibit low levels of 24,25(OH)₂D, exhibit hyperlipidemia, and develop atherosclerosis even on a standard rat chow diet. These findings suggest that 24,25(OH)₂D may be associated with atherosclerotic plaque formation (417), but the potential mechanism is unclear.

Several investigators have evaluated the possible association of calciotrophic hormones in the regulation of vascular calcification. Watson et al. (418) evaluated serum 1,25-(OH)₂D₃ and PTH levels in two populations of subjects: 153 asymptomatic individuals with at least a 5% risk of developing CHD within 4 yr, and 13 patients with familial hypercholesterolemia, most of them with documented CHD. In both groups, there was a negative inverse correlation of serum 1,25-(OH)₂D₃ with coronary artery calcification. Neither serum osteocalcin nor PTH was associated with coronary calcification in either group (418). A larger analysis of 283 asymptomatic subjects with risk factors for CHD reported that serum 1,25-(OH)₂D₃ independently and inversely predicted coronary calcification quantity measured by CT (419). A small preliminary study of 50 patients undergoing angiography failed to find any relationship between 1,25-(OH)₂D₃ and coronary calcification measured by CT (420), probably as a result of limited power to detect differences. However, differences in patient characteristics or methodologies used in these studies may also have contributed to the discordant results obtained.

It is not clear how serum 1,25-(OH)₂D₃ may be inversely related to coronary calcification. One explanation is that 1,25-(OH)₂D₃ inhibits arterial calcification by promoting the development and function of putative arterial OLCs (140, 141). Additionally, 1,25-(OH)₂D₃ may inhibit mineralization by suppressing vitamin D response elements in the promoter regions of the genes encoding PTH-related peptide, collagen I, and BSP. On the other hand, it is possible that serum measurements of 1,25-(OH)₂D₃ are not an accurate reflection of the effects of vitamin D within plaque microenvironments, because many factors such as clearance, synthesis, transport, and distribution of vitamin D all can affect serum levels of the hormone. Nevertheless, the potential relationship of 1,25-(OH)₂D₃ with arterial calcification is intriguing and warrants further investigation.

B. Estrogen
Epidemiological studies have clearly indicated that CHD rarely affects premenopausal women and that the gender protection is lost after menopause as incidence of CHD approaches that of men (421, 422). Moreover, the risk of CHD among premenopausal women who had undergone bilateral oophorectomy is more than double the risk among those who did not (423). These observations strongly suggest that endogenous estrogen possesses cardioprotective properties. Earlier observational studies have consistently suggested that hormone replacement therapy in postmenopausal women reduced the risk of coronary events by half (424, 425, 426). However, more recent randomized, placebo-controlled trials have shown no decrease or even an increase in the risk of CHD events in the first year of treatment with conjugated equine estrogen and medroxy-progesterone acetate (427, 428, 429, 430, 431). Despite such controversy about the possible benefits of estrogen therapy in clinical settings, a number of animal studies have supported the antiatherogenic effects of estrogen (recently reviewed in Refs. 432 and 433). Antiatherogenic effects of estrogen have been observed in studies using various models such as diet-induced hypercholesterolemic mice, rabbits, and monkeys, and gene knockout animals including apoe–/– (434, 435) and LDL receptor (ldlr) –/– mice (436). Animal studies have several advantages over clinical trials: bias can be greatly reduced through near-complete randomization; direct histological and molecular analysis of vascular tissues allows better elucidation of mechanisms; and experimental animals have much more uniform genetic backgrounds, thus minimizing genetic heterogeneity as a potential source of spurious results or error variance. It therefore seems reasonable to conclude that estrogen has atheroprotective effects at least under certain conditions, although clinical benefits of combination hormone therapy on CHD are still controversial.

Although its lipid-lowering effects have long been recognized and were previously considered to play a major role in protection from atherogenesis, recent evidence suggests that estrogen actions on lipid metabolism explain only about one third of its antiatherogenic effects (437, 438). Furthermore, the antiatherogenic effects of estrogen do not correlate with lipid-lowering effects in most animal studies (432). It therefore seems likely that the antiatherogenic effects of estrogen are in large part attributable to direct actions on the vascular wall (439), and additionally, to indirect systemic effects on coagulation and fibrinolytic systems, antioxidant systems, and production of vasoactive molecules (440) and proinflammatory cytokines (441).

Of particular relevance is activation of endothelial nitric oxide synthase (eNOS). eNOS is the principal source of nitric oxide (NO) in the vascular wall, and antiatherogenic effects of estrogen are considered to be mediated at least in part by increased availability of NO (440). Although effects of estrogen on eNOS are in part genomic (i.e., accompanied by an increase in the steady-state level of eNOS mRNA), nongenomic mechanisms of rapid activation of eNOS by estrogen have been of intense interest (reviewed in Ref.442). eNOS activation by estrogen is dependent on estrogen receptor (ER)- and involves various downstream signaling molecules such as inhibitory G protein, Akt/protein kinase B, extracellular signal-related kinase, and intracellular calcium in the EC caveolae (442). Nevertheless, the interaction of estrogen, eNOS activation, and atherogenesis in vivo remains poorly understood. It is intriguing that NO is also a potent regulator of bone metabolism (443) and that eNOS–/– mice exhibit defective bone formation and anabolic responses to exogenous estrogen (444, 445). This suggests that eNOS may also be involved in the pathogenesis of postmenopausal bone loss.

Postmenopausal osteoporosis is characterized by high bone turnover that is primarily caused by enhanced osteoclastic bone resorption coupled to activation of osteoblastic bone formation. Mechanisms of enhanced bone resorption with estrogen withdrawal have been extensively studied, and evidence supports critical roles for proinflammatory cytokines such as IL-6, IL-1, and TNF- (reviewed in Refs. 441, 446 , and 447). IL-6 (448, 449) and TNF- (450, 451) are direct ER targets of transcriptional repression, and IL-1 expression is known to be up-regulated by TNF- (452). As would be predicted by these findings, bone loss after ovariectomy in mice is indeed prevented by interfering with the action of each: deletion of IL-6 gene in knockout mice (453); loss of IL-1 action by deletion of type I IL-1 receptor gene in knockout mice (454) or treatment with soluble receptor IL-1ra (455); or blockage of TNF- action by treatment with TNF-binding protein (456) or transgenic overexpression of soluble TNF receptor (457). Induction of these proinflammatory cytokines upon estrogen withdrawal also appears relevant to atherogenesis, because atherosclerosis itself is an inflammatory process (6, 7, 8, 9, 14, 15, 16, 19, 297, 458, 459, 460) (see also Section II and references therein). All three cytokines above are produced in vascular tissues (441), and their roles in atherogenesis are supported by in vivo experiments similar to those described above for postmenopausal osteoporosis. However, the precise nature of their contribution to the antiatherogenic effects of estrogen remains controversial (461, 462, 463).

Association of estrogen status specifically with arterial calcification has been suggested by several clinical studies (464, 465, 466), but the direct impact of estrogen replacement on arterial calcification in postmenopausal women has not yet been tested. From the viewpoint that arterial calcification is formed through a mechanism similar to bone formation and may involve the opposing action of OLCs similar to bone resorption (140, 141), a number of estrogen targets participating in bone metabolism and/or postmenopausal osteoporosis could play a role in arterial calcification as well (Table 2). The association of estrogen status and calcium content of atherosclerotic plaques was examined in coronary arteries obtained from pre- and postmenopausal women at the time of autopsy. Estrogen status independently correlated with coronary calcium, plaque burden, and the proportion of calcium to plaque in women, regardless of whether they were pre- or postmenopausal. These associations were not explained by differences in known CHD risk factors or other possible confounding variables, such as osteoporosis and socioeconomic status. Intriguingly, estrogen use in postmenopausal women was associated with a significantly lower calcium-to-plaque ratio, suggesting that calcification and plaque formation may progress at different rates and that estrogen may differentially affect atherosclerosis and calcification (466).

In summary, substantial evidence indicates that estrogen has antiatherogenic activity, but the clinical efficacy and cardiovascular consequences of hormone replacement therapy in postmenopausal women remains controversial. A number of common estrogen targets may underlie the pathogenesis of postmenopausal osteoporosis, atherosclerosis, and possibly arterial calcification as well. However, only a weak association between osteoporosis and arterial calcification has been suggested, and a direct causal relationship has not been directly tested. If arterial calcification is a similar process to bone formation and resorption, the paradox that the vascular system calcifies as bone loses mineral (478) remains an intriguing dilemma of considerable clinical relevance that has yet to be explained.

C. Pharmaceutical agents that regulate calcification
The class of drugs called the bisphosphonates or diphosphonates were originally developed in 1968 to slow bone turnover for the treatment of metabolic bone diseases such as Paget’s disease (479, 480, 481, 482). These compounds were discovered because plasma and urine contain inorganic pyrophosphate compounds that inhibit calcium phosphate precipitation. Pyrophosphates bind avidly to calcium phosphate and impair both the precipitation and dissolution of calcium phosphate crystals. Pyrophosphates inhibit calcification in vivo, and ectopic calcification is prevented by parenteral administration of the compounds. Overall, these compounds may regulate both calcification and decalcification, depending on their concentration and activity.

Bisphosphonates are pyrophosphate analogs that contain a carbon atom instead of an oxygen atom. Bisphosphonates inhibit formation and aggregation of calcium oxalate crystals and avidly bind to calcium phosphate crystals, inhibiting their growth, aggregation, and dissolution. The main effect of pharmacologically active bisphosphonate is to inhibit bone resorption. Bisphosphonates inhibit the formation of pits by isolated osteoclasts cultured on mineralized substrata. In growing rats, bisphosphonates block the degradation of both primary and secondary trabeculae, arresting the modeling and remodeling that usually occurs in bone.

Like pyrophosphate, bisphosphonates efficiently inhibit calcification in vivo. They prevent experimentally induced calcification of many soft tissues such as arteries, kidneys, and skin and are active when administrated orally. In arteries, the administration of bisphosphonates decreases not only mineral deposition, but also accumulation of cholesterol, elastin, and collagen. One of the first-generation bisphosphonates, etidronate, inhibits bioprosthetic heart valve calcification, the formation of urinary stones, formation of dental calculus, and normal mineralization of bone. In the skeleton, the impairment of mineralization can lead to fractures and impairment of fracture healing. At high doses, in skeletal tissue, the inhibition of mineralization may result in osteomalacia or rickets. It is precisely this effect that results in the suppression of atherogenesis in animal models (62, 483). It is notable that these early compounds not only inhibited calcification, but also reduced collagen and elastin synthesis in arterial tissue. The newer generation bisphosphonates, ibandronate and alendronate, also inhibit arterial calcification in experimental animals (52, 69). Newer bisphosphonates that are under development have cholesterol-lowering properties and antiatherosclerotic effects as well.

Statins inhibit hydroxymethylglutaryl coenzyme A reductase, the rate-limiting step in cholesterol synthesis, and reduce coronary artery disease (CAD) morbidity and mortality (17). Statins decrease vascular calcification in direct proportion to their ability to lower LDL cholesterol (484), but also have other beneficial cardiovascular effects that appear unrelated to their favorable effects on lipoprotein profiles (17). Bisphosphonates and statins seem to have similar effects on both bone remodeling and lipid metabolism. The aminobisphosphonates act in the same metabolic pathway by inhibiting farnesyl diphosphate synthase, which is downstream of hydroxymethylglutaryl coenzyme A reductase. These bisphosphonates also have lipid-lowering effects (485). The mechanism of bisphosphonate inhibition of bone resorption is through a pathway common to macrophages, which, like osteoclasts, undergo apoptosis on exposure to the compound. Select statins, such as mevastatin, also cause apoptosis of macrophages, an effect that is prevented by the addition of mevalonate (486). The action of one of the commonly used bisphosphonates on the market for the treatment of osteoporosis is also attenuated by the addition of mevalonate, suggesting similar mechanisms of action. The enzymes of the mevalonate pathway in osteoclast activity are regulated by prenylation of guanosine triphosphate-binding proteins that have multiple diverse cellular functions (17).

Other studies further suggest that statins and bisphosphonates exert similar effects in bone, lipid metabolism, and perhaps vascular tissues. For example, statins increased new bone formation in vitro through activation of the BMP-2 promoter (487, 488). Oral administration of simvastatin increased bone volume and the rate of bone formation in ovariectomized rats (489). However, the ability to stimulate BMP-2 expression is not a general feature common to all statins. Several clinical case-control or retrospective studies using medical records suggested that there was a reduction in fracture rates in individuals receiving statins (490, 491). Some studies suggested reduction in fracture as large as 50%, which is comparable to that seen with bisphosphonates. In contrast, statins that do not increase BMP-2 expression have not been associated with changes in fracture rates. Overall, there is intriguing evidence and a plausible cellular mechanism for commonality between the effects of statins and the bisphosphonates on both bone and lipid metabolism. Longer-term prospective clinical studies as well as carefully designed animal studies are needed to enhance our understanding of these intriguing interactions. At present, there is very limited evidence that supports a role for these pharmacological agents in the mechanism of arterial calcification, and this will be a fruitful area for future investigations.

D. Role of lipids
Because statins alter bone remodeling, it has been hypothesized that lipid metabolism influences the development of arterial calcification (150, 151). Tissue culture studies have shown that vascular or mesenchymal cells can be induced to form mineralized nodules, and this process is facilitated by oxidative stress (492) and leptin (493) and inhibited by high-density lipoprotein (HDL) (494, 495). However, bone marrow stromal cells isolated from C57BL/6 mice fed a high-fat diet failed to differentiate into osteoblasts when cultured in vivo (496), and minimally oxidized LDL inhibited in vitro mineralization (497). One possible interpretation of these results is that specific oxidized lipids differentially affect calcification in both bone and artery. Also, as already noted, statins affect not only bone formation but also bone resorption, and the net effect on arterial calcification may not be straightforward. Whatever the explanation for these cell culture studies, the relevance to in vivo conditions is uncertain. Clinical studies of arterial calcification have consistently shown that serum levels of LDL cholesterol are directly related and HDL cholesterol is inversely related to the prevalence and extent of coronary artery calcification measured with CT (21, 22, 23). Although suggestive, it cannot be concluded that lipoproteins had any direct effect on calcification per se. Coronary calcification is a surrogate measure of coronary atherosclerosis, and histologically there is good correlation between the quantities of atherosclerosis and calcification (143). But serum LDL cholesterol is a well-established risk factor for CAD (12, 13, 17), and can thus be considered a surrogate measure of atherosclerosis as well (498, 499, 500, 501, 502, 503). To test the hypothesis that serum lipoproteins participate in arterial calcification will require controlled experiments designed to test how levels of LDL and HDL cholesterol affect calcification independently of their effects on atherosclerosis.

Recent studies have revealed a central link between the roles of leptin in bone remodeling and lipid metabolism and also effects of leptin on bone formation that are mediated via the sympathetic nervous system (504, 505). Leptin is known to regulate fat metabolism and body weight by a hypothalamic mechanism (506, 507, 508, 509, 510). The influence of leptin on bone mass also involves hypothalamic mechanisms, but these are distinct from those involved in regulation of body weight and fat metabolism (505). It appears that leptin also influences bone mass through a direct effect by specific sympathetic ß2-adrenergic nerves on osteoblasts (505). There are several aspects of this model that remain controversial, however (510). Nevertheless, it will be interesting to determine how leptin might influence arterial calcification and how this might be altered by dyslipidemia and menopause.

	V. Genetic Determinants of Arterial Calcification

Top Abstract I. Introduction: Clinical... II. Types of Arterial... III. Molecular Mechanisms of... IV. Endocrine, Pharmacological,... V. Genetic Determinants of... VI. Conclusions References

 
The use of genetically altered animal models has proven invaluable in unraveling the genetic components contributing to atherosclerosis (511, 512, 513), but significant progress has emerged from clinical, epidemiological, and information analysis approaches as well (513, 514, 515, 516, 517, 518, 519). Nevertheless, there are distinct indications that our understanding of the genetic determinants of atherosclerosis and arterial calcification is still quite limited. For example, risk factors for CHD have a strong genetic component, and risk factors predict clinical CHD, but predictive power is very limited. A significant proportion of CHD occurs in individuals with unremarkable risk factor profiles, and conversely, many who have elevated CHD risk never suffer any clinical manifestation of the disease. Furthermore, some of the genetic components of CHD are independent of the effects of risk factors (520, 521). Several genetic loci for myocardial infarction have been identified (522, 523), some of which are in chromosomal regions that do not harbor any known genes associated with atherosclerosis or risk factors (524).

Data obtained so far are limited, but there also appears to be a strong genetic component for the prevalence and extent of arterial calcification that is at least partially independent of genetic determinants of atherogenesis (525). Histopathological (143, 526) and in vivo intravascular ultrasound (527, 528) studies indicate a close relationship between measures of atherosclerosis and coronary artery calcification. Because of the high correlation between atherosclerotic plaque and intimal calcification, it might seem reasonable to presume that atherosclerotic calcification is a simple consequence of plaque development. However, three lines of direct and indirect evidence indicate that this may be an overly simplified view:

1) Important determinants of coronary calcification prevalence and quantity include known CAD risk factors such as male gender, age, smoking status, cholesterol levels, high blood pressure, and body mass (529, 530, 531, 532, 533, 534, 535, 536), but these account for only about 40% of the observed interindividual variability in coronary artery calcium quantity, and at least another 40% of the variability is derived from largely unknown genetic factors (537).

2) In a recent genomewide linkage analysis of sib pairs who also underwent coronary calcium assessment with CT, Lange et al. (538) reported that two chromosomal loci (6p21.3, maximum LOD score, 2.22; and 10q21.3, maximum LOD score, 3.24) may harbor genes that are important determinants of arterial calcification. These findings have been corroborated by a report on parent-offspring and sibling pairs from the Framingham Heart Study, which found that after adjustment for coronary risk factors, half of the observed variance in aortic calcification was due to the combined effects of genetic factors (539).

3) Coronary arterial calcification appears to segregate according to racial origins. Specifically, several studies report that blacks exhibit significantly less coronary calcification than whites, even after adjusting for the effects of coronary risk factors and other ethnic differences (540, 541, 542). Histopathological studies also indicate that blacks manifest both a lower prevalence and quantity of coronary calcification (543, 544, 545). It is not likely that these findings can be explained by differences in the extent of atherosclerosis, because despite having lower amounts of coronary calcium, blacks nevertheless suffer greater numbers of coronary events (541). Furthermore, after adjusting for risk factors, the amount of carotid artery (546, 547) and cerebrovascular (548) atherosclerosis is similar in blacks and whites, and blacks have greater amounts of common carotid atherosclerosis (546). Collectively, these studies provide indirect evidence consistent with the interpretation that there is some degree of segregation of the arterial calcification phenotype according to race and that this is partially independent of genetic determinants of atherosclerosis.

Studies examining the role of specific candidate genes in arterial calcification are limited. Nine specific genes have thus far been linked to arterial calcification in humans: angiotensin I-converting enzyme (ACE), apo E, E-selectin, MMP-3, MGP, CC chemokine receptor 2 (CCR2), ER, epoxide hydrolase, and ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1). In addition, there are indications that OPG may play a role in atherosclerosis and, potentially, in arterial calcification, but this possibility has not yet been investigated.

A. Angiotensin-converting enzyme
The renin-angiotensin system plays a key role in the etiology of several cardiovascular pathologies, including atherosclerosis (for reviews, see Refs. 549 and 550). Several polymorphisms in the human ACE gene, including the M235T and insertion/deletion (I/D) polymorphisms, have been identified (551). The association of the ACE I/D polymorphism with CAD has been extensively studied (552, 553, 554) and is associated with myocardial infarction (555, 556, 557, 558) and CAD (558, 559, 560, 561). Data from a prospective randomized trial (562) and a case-control study (563) failed to confirm these findings, and therefore, the role of the ACE I/D polymorphism in atherosclerotic disease remains uncertain. However, evidence from a study of patients with documented CAD and coronary calcification suggests a role for the ACE I/D polymorphism in arterial plaque calcification. In a study of 146 patients undergoing interventional coronary procedures, Pfohl et al. (564) assessed coronary artery calcification with intravascular ultrasound and analyzed genomic DNA for ACE I/D polymorphisms. After correction for coronary risk factors that are themselves determinants of atherosclerotic calcification (529, 530, 531, 532, 533, 534, 535, 536), the DD genotype was significantly associated with more calcified lesions, as well as greater circumferential calcific involvement in the target lesion segment (564). Although the sample population was comparatively small and biased, the frequencies of the ACE genotypes were similar to those reported by larger studies from both Europe (555, 563) and the United States (562), and the distribution of ACE genotypes approximated Hardy-Weinberg equilibria.

B. Apolipoprotein E
Apo E participates in extracellular cholesterol transport (565, 566, 567, 568) and has important homeostatic functions in diverse metabolic pathways (568). Abnormalities in apo E have been implicated in a number of pathologies, including atherosclerosis (569, 570, 571, 572). Studies in mice report that genetic deficiency of apo E leads to severe hypercholesterolemia, atherosclerosis (272, 273), and plaque calcification (254, 255, 256). The gene encoding apo E generates three major isoforms (apo4, apo3, and apo2). A number of studies have linked the 4 variant of apo E to both the presence and clinical sequelae of coronary atherosclerosis (573, 574, 575, 576, 577). Kardia et al. (578) assessed the relationship between apo E genotype and coronary calcification determined by CT scanning in 329 asymptomatic subjects. Associations between variations in risk factors and variations in the probability of coronary calcification being present appeared to be dependent on apo E genotype, but adding apo E genotype to risk factor logistic algorithms failed to improve predictive power. One possible interpretation is that apo E genotype may modulate the contribution of known coronary risk factors to the prevalence and amount of arterial calcification. However, further studies will be needed to better elucidate the possible role of apo E genotype as a genetic determinant of arterial calcification.

C. E-selectin
Increased expression of selectins by arterial ECs is one of the early events of atherosclerosis (6, 7, 8, 9, 19). Selectins are a family of adhesion molecules that mediate tethering and rolling interactions between leukocyte and hematopoietic progenitors and ECs. E-selectin, also known as endothelial-leukocyte adhesion molecule 1, is one such adhesion factor expressed by ECs (579, 580, 581, 582), and this protein is recognized by cells expressing its cognate ligand CD44 (E-selectin ligand) on their cell surface (583, 584). E-selectin is important in the homing of hematopoietic progenitors to bone (585, 586). Bone marrow vascular cells express E-selectin (587), which appears to aid in homing of osteoclast progenitors circulating in the mononuclear fraction (230, 231, 232, 233, 234). E-selectin may thus represent a common element between vascular inflammation, such as occurs in atherosclerosis, and homing of bone cell progenitors. It has been proposed that E-selectin-mediated homing of bone cell progenitors to arterial ECs may play a role in plaque calcification (140), but this possibility has not yet been investigated and therefore remains speculative.

A mutation in the gene encoding E-selectin results in a substitution of arginine for serine 128 within the epidermal growth factor-like domain of the mature protein (588). This polymorphism, known as S128R, significantly alters ligand recognition and binding by increasing the affinity for other ligands, thus decreasing binding specificity (589). Several studies suggest an association of the S128R polymorphism with greater severity of atherosclerosis (590, 591). One study has examined the relationship of the S128R E-selectin polymorphism with coronary artery calcification. Ellsworth et al. (592) compared the incidence of this polymorphism with coronary calcification using CT quantification in 608 asymptomatic low-risk patients. They reported a significant association between the E128R polymorphism and both the presence and quantity of coronary calcification in women 50 yr of age or younger, after adjusting for the effects of coronary risk factors. However, no relationship was observed for men or older women. An important caveat in the interpretation of these results is that estrogen inhibits expression of adhesion molecules in ECs, and hormone replacement therapy in postmenopausal women results in a reduction of circulating levels of adhesion molecules including E-selectin (439, 593). Therefore, it is unclear whether the S128R polymorphism is independently related to coronary calcification (at least in some women), or whether the association observed was a secondary effect of variability in estrogen levels and their effects on adhesion molecules. Further studies will be required to more completely elucidate a possible role of E-selectin polymorphisms in determining atherosclerotic calcification.

D. Matrix metalloproteinase-3 (MMP-3)
MMPs comprise a family of over two dozen zinc-dependent endopeptidases that are largely responsible for homeostatic remodeling of extracellular matrix elements during embryo development, morphogenesis, and also in adult tissues as part of normal tissue repair processes (594). Proteolytic activities of MMPs are blocked by a number of endogenous inhibitors, notably the family of four TIMPs (595, 596). Imbalances in the relative expression and activities of MMPs and their inhibitors have been implicated in diverse pathologies, including cardiovascular disorders (594, 597, 598, 599, 600). Several MMPs play important roles in bone formation and remodeling (601, 602, 603) (Fig. 6). MMP-3 (also known as stromelysin 1) activity has been implicated in both bone formation (604, 605) and resorption (606, 607, 608, 609, 610, 611, 612, 613), progenitor and stem cell mobilization from bone marrow to permissive vascular niches (614), and bone-related pathologies (609, 610, 611, 612, 613, 615). Because MMP-3 participates in many bone-related processes, it might be speculated that it could also be involved in the mechanism of arterial calcification. Circumstantial observational evidence is at least consistent with this hypothesis: MMP-3 colocalizes with calcification in human carotid artery lesions (247). In addition, the MMP-3 promoter has several polymorphisms that have been associated with increased carotid artery intima/media thickness (616), restenosis following angioplasty (617), and stable angina pectoris (618). A report from the Helsinki Sudden Death Study found that in men over the age of 53 yr, mean calcified area of lesions assessed postmortem was significantly associated with an MMP-3 polymorphism (619). Subjects with high promoter activity genotypes (5A5A) had significantly larger calcified plaque areas after adjusting for coronary risk factors and the number of diseased arteries. However, this study did not fully control for the extent of atherosclerosis, and therefore it is uncertain whether the results may have been affected secondarily by the influence of the 5A5A promoter on atherogenesis.

E. Matrix Gla protein
The possible role of MGP in both medial and plaque calcification has been discussed above (see Sections III.A and IV.D), and there is evidence that MGP polymorphisms are associated with atherosclerosis and intimal calcification as well. At least eight polymorphisms have been identified (620, 621, 622, 623). Herrmann et al. (620) assessed carotid and femoral artery atherosclerosis and calcification and determined the relationships among MGP polymorphisms. Two polymorphisms were related to femoral artery calcification, but none was associated with carotid artery calcification. In a case-control substudy, there were no relationships among MGP polymorphisms and myocardial infarction, except in one subgroup (620). This study therefore provides only weak evidence for an independent genetic contribution of MGP polymorphisms to arterial calcification. Furthermore, the finding of an association of femoral but not carotid artery calcification with MGP genotype raises the possibility that any genetic relationship of these MGP polymorphisms with arterial calcification might be due to an association with Mönckeberg’s medial calcification, which is much more common in the femoral artery compared with the carotid artery.

An inherited human disorder, Keutel syndrome, is caused by a mutation that results in expression of a nonfunctional MGP product (391). Patients with Keutel syndrome manifest a constellation of bone and cartilage disorders and, curiously, pulmonary artery stenosis resulting in pulmonary hypertension (624, 625). This latter finding has not been reported to occur in MGP knockout mice, which develop massive medial arterial calcification (127, 128, 134). Conversely, however, no studies have examined the prevalence and extent of arterial calcification in these patients. It therefore appears that there are species differences in the contribution of MGP to arterial calcification, and the reason(s) will obviously require further investigation.

F. CC chemokine receptor 2
MCP-1 is a cytokine synthesized by arterial SMCs, macrophages, and ECs in response to inflammatory stimuli that plays an important role in early atherogenesis by providing a chemotactic signal for the recruitment of monocytes. MCP-1 effects are mediated via a seven-transmembrane G protein-coupled receptor, CCR2 (for reviews, see Refs. 308 and 626, 627, 628). Disruption of either MCP-1 (629) or CCR2 (630) in mice inhibits plaque formation in a dose-dependent fashion. In addition, MCP-1 may play a significant role in bone pathology and perhaps normal bone development and function. Osteoblasts in bone may (631) or may not (632) normally express MCP-1, but a number of studies indicate that induction of MCP-1 occurs in response to TGFß (632) and inflammatory stimuli, including infectious agents (633, 634, 635, 636, 637).

A number of polymorphisms in the gene encoding CCR2 have been described (638, 639, 640, 641, 642) that have been linked to conditions such as HIV infection, pulmonary sarcoidosis, inflammatory diseases, and myocardial infarction. One of these results in a substitution of Val to Ile at amino acid 64 in the full-length protein product and is thus known as the Ile64 variant. This polymorphism has been associated with reduced CCR2 function. Valdes et al. (643) recently measured coronary calcification with CT and compared calcium scores with CCR2 genotype in 672 first-degree relatives of patients with premature CAD (defined as documented CAD before age 60 in men and 70 in women). The Ile64 allele was associated with significantly less coronary artery calcification, independently of coronary risk factors. Interestingly, it has been reported that blacks have higher frequencies (0.15) (Refs. 612 and 613) of this allele than whites (0.07 to 0.10) (Refs. 607, 609, 611 , and 616). Thus, increased prevalence of this polymorphism among blacks may, at least in part, explain the decreased calcification reported in blacks (541, 542, 543, 544, 545, 546).

G. Estrogen receptor-
The role of ER variants in the development of atherosclerosis in men was recently explored in 300 white Finnish male autopsy specimens (644). Correlation of fatty streaks, fibrosis, calcification, and complicated lesions were related to the ER PvuII genotypes (P/P, P/p, and p/p). In men aged 53 yr and older, the mean areas of complicated lesions, calcification, and the presence of coronary thrombosis were significantly associated with the ER genotype, but this association was not present in younger men. After adjustment for age and body mass index in the older group, P/p and p/p genotypes had areas of complicated lesions that were 2- to 5-fold larger, respectively, compared with the p/p genotype. This interaction was true for the presence of calcium (P = 0.028) in the older group of men, but was nonsignificant for men younger than 53 yr of age. Men aged 53 yr or older had a 2-fold increase in the area of calcified lesions if they had the P/P or P/p genotype. Genotypic associations were not present for fibrosis, fatty streaks, or percentage of coronary narrowing, regardless of age. This study suggests that the ER polymorphism may mediate, in part, the action of estrogen on the arterial wall and may influence the process of calcification (644).

The discovery of a man with a null mutation in ER has provided additional insights into the relationship between the skeleton, vascular tissue, and estrogen action (645). Estrogen has significant effects on bone, lipid, and carbohydrate metabolism, and many of these factors resulted in skeletal deficiencies and premature atherosclerosis detected by coronary calcification. In both men and women, estrogen has critical roles in bone that include action in the pubertal growth spurt, in skeletal maturation, in the arrest of linear growth by fusion of the epiphyseal growth plate, in the accrual of peak bone mass, and in skeletal maintenance and repair. The patients with a null mutation in ER have high turnover osteoporosis without epiphyseal fusion. Lipid abnormalities included low serum concentrations of cholesterol, LDL-cholesterol, apo (a), and apo A1, but normal serum triglycerides, and premature atherosclerosis as manifest by increased coronary calcification on electron beam CT. Other vascular abnormalities included the absence of flow-mediated vasodilatation in brachial artery (endothelium-dependent vasodilatation). In contrast, there was an intact, rapid brachial vasodilatory response to sublingual estradiol and an increase in brachial artery flow velocity (a nongenomic action involving calcium-activated potassium channels in vascular SMCs) in this patient. This patient’s genetic abnormality indicated that the relationship between the skeleton and vascular tissue was complex but may be linked by the actions of estrogen on target tissues.

Of potential relevance, more than 10 cases of mutations in the gene encoding P450 aromatase have now been reported (646, 647, 648, 649, 650, 651, 652, 653, 654, 655, 656). Aromatase catalyzes the conversion of androgens to estrogen (657), is expressed by vascular ECs (658, 659) and SMCs (660), and has been detected in atherosclerotic plaques in human aortae (661). Data from murine models of atherosclerosis (ldlr–/–) indicate that testosterone may inhibit plaque development by aromatase-catalyzed conversion to estrogen (662). Relevance of these animal data to human atherosclerosis is suggested by the recent report that treatment of aromatase deficiency with estrogen replacement can cause regression of carotid atherosclerosis (646). Reports of patients with aromatase deficiency reveal an important role for aromatase in normal skeletal development, suggesting the possibility that aromatase expression and activity might influence arterial calcification. However, no studies to date have investigated the possibility that aromatase mutations or polymorphisms might be linked to development of plaque calcification.

H. Ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1)
ENPP1 (formerly known as plasma-cell differentiation antigen-1, or PC-1) is one of five related cell surface enzymes that participates in the degradation of nucleotides, releasing nucleoside 5'-monophosphate in the process (663, 664). Generation of the inorganic pyrophosphate in turn regulates a number of processes, including bone mineralization (665). ENPP1 is also expressed in various soft tissues including muscle, fat, liver, and kidney, although the role of ENPP1 in these tissues is unknown. Interestingly, ENPP1 suppresses insulin receptor tyrosine kinase activity, apparently through a direct interaction with the insulin receptor -subunit, and thus leads to insulin resistance (666, 667, 668, 669). A polymorphism in exon 4 that causes an amino acid change from lysine to glutamine at codon 121 (K121Q) appears to be associated with lower insulin sensitivity and increased risk of developing ESRD (649).

Rutsch et al. (670) recently reported that a number of loss-of-function mutations in the ENPP1 gene were present in eight of 11 kindreds with idiopathic infantile arterial calcification, a genetic disorder characterized by medial calcification and mineralization of the internal elastic fibers, and also myointimal proliferation leading to arterial stenosis. The strong association of ENPP1 loss-of-function mutations with arterial calcification suggests that decreased expression and/or function of ENPP1 may participate in medial calcification in other settings, particularly in diabetes. However, none of the mutations associated with idiopathic infantile arterial calcification involved the K121Q polymorphism that is associated with diabetic pathology. It is possible that ENPP1 polymorphisms might play a role in atherosclerotic calcification as well, but this has not yet been investigated.

A related human disorder called ossification of posterior longitudinal ligament of the spine is a myelopathy disorder among Japanese and other Asians characterized by ectopic bone formation in the paravertebral ligament. This condition appears to occur by an endochondral mechanism and can be caused by a number of metabolic and genetic abnormalities, including mutations in ENPP1 (671, 672, 673). The tiptoe-walking mouse mutation (ttw) is a murine homolog of this disorder (674, 675, 676). Vascular calcification, whether medial or in the setting of atherosclerosis, has not been investigated in either the human disorder or the ttw mouse. It would be interesting to determine whether ttw mice that are rendered atheroslerosis-prone would exhibit an increased amount of intimal plaque calcification.

I. Epoxide hydrolase
Arachadonic acid metabolism produces an array of epoxyeicosatrienoic acid (EETs) lipid products. EETs, produced by vascular endothelium via the cytochrome P450 system (677, 678), are antiinflammatory vasodilators (679) with numerous vascular effects, including regulation of platelet function (680), SMC proliferation (681), and vascular permeability and tone (682, 683). Degradation of EETs into diols (dihydroeicosatrienoic acids) is catalyzed by the ubiquitously expressed enzyme epoxide hydrolase, which plays a key role in regulating EET levels. Seven polymorphisms have been identified in the coding region of the epoxide hydrolase gene, of which five are silent mutations (684). The two other variants result in a substitution of glutamine for arginine or an unusual in-frame trinucleotide insertion at the same position in the genomic sequence, resulting in two sequential arginine residues rather than one in the protein product. The replacement of arginine by glutamine results in a protein with less activity than the wild type, and the arginine insertion markedly decreases enzyme activity (684). Fornage et al. (685) recently reported that after adjusting for coronary risk factors, presence of at least one copy of the substitution polymorphism was associated with a 2-fold greater risk (95% CI, 1.1 to 2.9) of having at least some coronary artery calcification in black subjects (compared with subjects with two normal alleles) and was also related to the quantity of calcification measured by CT. However, there was no significant association between the presence of this polymorphism and the probability of a white subject having coronary calcification. These findings could be interpreted as supporting the hypothesis that there are ethnic differences in the pathobiological determinants of coronary calcification (540, 541, 542, 686). However, it is also possible that because atherosclerosis is multifactorial, there could be a differential effect of the arginine to glutamine substitution depending on the genetic context. For example, the polymorphism might have a differential impact on other risk factors that themselves exhibit ethnic variability. Additional studies are warranted to shed more light on these issues.

J. Osteoprotegerin
Genetically altered mice unable to express functional OPG demonstrate osteoporosis and, surprisingly, arterial calcification in the aorta and large arteries (336, 341). Serum OPG levels are elevated in patients with diabetes (687, 688). Furthermore, after adjusting for the effects of age, OPG levels are associated with all-cause mortality (OR, 1.4/SD; 95% CI, 1.2 to 1.8) and cardiovascular mortality (OR, 1.4; 95% CI, 1.1 to 1.8), and these effects were not confounded by diabetes (687). Jono et al. (689) reported similar findings in 201 patients with stable angina undergoing angiography. In this study, using multivariate logistic regression, the OR were somewhat greater (OR, 5.2; 95% CI, 1.7 to 16.0). In another angiographic study of 522 age-matched men, Schoppet et al. (688) also reported that OPG serum levels are associated with the presence and severity of CAD and are increased in elderly men and patients with diabetes mellitus. Preliminary data from these same investigators suggest that serum levels of free RANKL (i.e., not complexed to OPG) were significantly lower in patients with CAD, but were not correlated with the severity of CAD (690). Recently, a thymine/cytosine polymorphism was identified that was associated with increased carotid artery intima-media thickness in patients homozygous for this base pair substitution (691). Unfortunately, arterial calcification was not reported in these studies. Another mutation in the gene encoding OPG results in a juvenile idiopathic phosphatasia phenotype characterized by progressive deformities of long bones, osteopenia, fractures, kyphosis, and acetabular protrusio, coupled with extremely rapid bone turnover, with markedly increased indices of both bone resorption and formation (692, 693). No studies have yet investigated whether this rare mutation affects development of atherosclerotic plaque calcification. Taken together, these studies suggest the need for additional investigation into possible genetic links between OPG, RANK, and RANKL and the development of arterial calcification.

K. Other human genetic disorders and arterial calcification
Werner syndrome is a rare autosomal recessive disorder (694, 695, 696) characterized by premature aging, bone and connective tissue pathologies (697, 698), and ectopic calcification in a number of soft tissue locations including the cornea (699), heart valves (700), and large arteries (701, 702). The gene mutation causing human Werner syndrome, WRN, encodes a 180-kDa nuclear protein that contains both exonuclease and helicase functional domains. The type of arterial calcification is partly medial and partly intimal, because premature aging is also accompanied by rapidly developing atherosclerosis and other nonatherosclerotic vascular pathologies. Most patients die of myocardial infarction before the age of 50. Castro et al. (703) analyzed the frequencies of two polymorphisms at the WRN locus (1074Leu/Phe and 1367Cys/Arg) in Finnish and Mexican patients and observed an age-dependent decline of 1074Phe/Phe genotype. There was a trend toward gene dose-dependent associations of the 1074Phe (direct) and the 1367Arg/Arg alleles (inverse) with coronary stenosis, but these tendencies did not reach statistical significance. Unfortunately, these investigators did not assess arterial calcification. Because of the very high prevalence and extent of vascular calcification at an early age in Werner syndrome patients, it remains possible that the WRN gene could be a determinant of arterial calcification. Additional studies are needed to test this hypothesis.

The active model of arterial calcification implies that osteoblast-like cells develop from progenitors, and the finding that BMPs are expressed in arteries and colocalize with calcified regions of plaque has led to the notion that BMP signaling is involved in this process. Also, mice unable to express Smad6, a downstream cytoplasmic inhibitor of BMP signaling, demonstrate marked arterial calcification (704). As with other genotypes such as MGP–/– and OPG–/–, these mice are not prone to atherosclerosis, and it therefore remains uncertain whether this phenotype is relevant to plaque calcification. Furthermore, the possibility that BMP signaling is involved in plaque calcification has not been directly tested in vivo. It was recently reported that both human and mouse aortae express BMP receptors (BMPRs) and components of the BMP signaling pathway (705). However, the function of BMP signaling in arteries remains unknown. Whether or not BMP signaling participates in atherosclerosis or arterial calcification is an untested hypothesis and remains speculative. Expression of BMPRs has also been reported in pulmonary arteries, and mutations in BMPR-II cause primary pulmonary hypertension in humans (706, 707, 708, 709, 710, 711), but pulmonary arteries are not susceptible to atherosclerosis. Although several BMP ligands have been found in arteries, it is possible that expression of BMPs results from calcification rather than causes it. It is also conceivable that arterial expression of BMPs is a concomitant that is not directly involved in plaque calcification. Obviously, considerable additional investigation using in vivo models will be required to fully elucidate which of these possibilities may be relevant, and it will also be important to determine whether patients with mutations in BMPR-II and other components of the BMP signaling pathway demonstrate greater or lesser quantities of arterial plaque calcification.

	VI. Conclusions

Top Abstract I. Introduction: Clinical... II. Types of Arterial... III. Molecular Mechanisms of... IV. Endocrine, Pharmacological,... V. Genetic Determinants of... VI. Conclusions References

 
It is evident that there is much to be learned regarding the genetic contribution to the observed phenotypic variability of atherosclerosis and, particularly, arterial plaque calcification. Although arterial calcification has been studied extensively, very few studies (256) have directly tested molecular and genetic mechanisms of atherosclerotic plaque calcification in a whole animal model. Future investigations should be focused less on observation and more on mechanistic testing and experimental interventions. Among the many questions that require answers then, the more urgent ones include: what are the molecular and genetic mechanisms that affect plaque calcification, and how do these differ from those causing atherosclerosis? To what extent do polymorphisms in relevant genes account for the observed variability in the phenotypic expression of these pathologies that cannot be accounted for by risk factors alone, and what are their frequencies in populations? How do genetic differences alter the response to treatment strategies? What is the nature of the apparent differences between genetically altered animal models and human mutational equivalents? And how do known mutations causing inherited human bone disorders affect plaque calcification? Unraveling the genetic determinants of both atherosclerosis and intimal plaque calcification provides fertile ground for investigations that will significantly enhance our understanding of vascular disease.

	Acknowledgments

 
We are grateful to Dr. Steven L. Goldberg (Division of Cardiology, University of Washington, Seattle WA), Dr. Matthew J. Budoff (Division of Cardiology, Harbor UCLA Medical Center, Torrance CA), and Dr. Richard L. Kurtz (Department of Physics and Astronomy, Louisiana State University, New Orleans, LA) for kindly supplying unpublished images for the figures; Ruth Kiefer (Mayo Foundation, Rochester, MN) for expert secretarial support; Kolja Wawrowsky, M.S., and Hiroyasu Uzui, M.D., Ph.D. (Cedars-Sinai Medical Center, Los Angeles, CA) for technical assistance; and Ejaz Ahmed, M.D., Ph.D., for reviewing the manuscript. We are particularly thankful to Richard Clyde French for so freely providing ongoing assistance that made this work possible. We dedicate this work to the late Professor Michael J. Davies (712 ), whose seminal histopathological and clinical correlative investigations established lofty benchmarks of excellence, revolutionized our understanding of cardiovascular pathologies, and eloquently and profoundly impacted us all.

	Footnotes

 
This work was supported by grants from the Philip Morris USA, Inc. (to T.B.R.), the National Heart, Lung, and Blood Institute (HL51980 and HL58555 to T.B.R.; HL51736 to L.A.F., and 7RO1-HL43277-02 to R.C.D.), the National Center for Research Resources (K24RR017593-01 to L.A.F.), the National Institutes of Health, the Mayo Foundation, and the Mirisch Foundation, United Hostesses Charities, the Eisner Foundation, the Grand Foundation, the Ornest Family Foundation, and the Heart Fund at Cedars-Sinai Medical Center.

Abbreviations: ACE, Angiotensin-converting enzyme; AHSG, ₂-Heremans-Schmid glycoprotein; apo, apolipoprotein; bcl-2, B cell chronic leukemia-2; BGP, bone Gla protein; BMP, bone morphogenetic protein; BMPR, BMP receptor; BSP, bone sialoprotein; CA-II, carbonic anhydrase II; CAD, coronary artery disease; cat K, cathepsin K; Cbfa1, core-binding factor 1; CBFß, core-binding factor ß; CCR2, CC chemokine receptor 2; c-fms, cellular-feline McDonough sarcoma protooncogene; CHD, coronary heart disease; CI, confidence interval; CT, computed tomography; CTR, calcitonin receptor; CVC, calcifying vascular cell; Dlx5, Distal-less5; dpp, decapentaplegic; EC, endothelial cell; EET, epoxyeicosatrienoic acid; eNOS, endothelial NO synthase; ENPP1, ecto-nucleotide pyrophosphatase/phosphodiesterase 1; ER, estrogen receptor; ESRD, end-stage renal disease; HDL, high-density lipoprotein; I/D, insertion/deletion; K1, phylloquinone; LDL, low-density lipoprotein; MCP-1, monocyte chemotactic protein-1; M-CSF, macrophage colony-stimulating factor; MGP, matrix Gla protein; MK, menaquinone; MMP, matrix metalloproteinase; MPC, mononuclear phagocytic cell; Msx2, muscle segment homeobox 2; NO, nitric oxide; 1,25-(OH)₂D₃, 1,25-dihydroxyvitamin D₃; 24,25-(OH)₂D, 24,25-dihydroxyvitamin D; OLC, osteoclast-like cell; OPG, osteoprotegerin; OPN, osteopontin; OR, odds ratio(s); Osx, osterix; RANK, receptor activator of nuclear factor-B; RANKL, RANK ligand; Smad, mammalian homolog of mothers against dpp; SMC, smooth muscle cell; TIMP, tissue inhibitor of MMP; TRAF, TNF receptor-associated factor; TRAP, tartrate-resistant acid phosphatase; WRN, gene mutation causing human Werner syndrome.

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