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Inflammation and the Osteogenic Regulation of Vascular Calcification

2010; Lippincott Williams & Wilkins; Volume: 55; Issue: 3 Linguagem: Inglês

10.1161/hypertensionaha.109.134205

1524-4563

Jian-Su Shao, Su‐Li Cheng, Justin S. Sadhu, Dwight A. Towler,

Bone health and treatments

HomeHypertensionVol. 55, No. 3Inflammation and the Osteogenic Regulation of Vascular Calcification Free AccessReview ArticlePDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessReview ArticlePDF/EPUBInflammation and the Osteogenic Regulation of Vascular CalcificationA Review and Perspective Jian-Su Shao, Su-Li Cheng, Justin Sadhu and Dwight A. Towler Jian-Su ShaoJian-Su Shao From the Department of Medicine, Washington University in St. Louis, St. Louis, Mo. , Su-Li ChengSu-Li Cheng From the Department of Medicine, Washington University in St. Louis, St. Louis, Mo. , Justin SadhuJustin Sadhu From the Department of Medicine, Washington University in St. Louis, St. Louis, Mo. and Dwight A. TowlerDwight A. Towler From the Department of Medicine, Washington University in St. Louis, St. Louis, Mo. Originally published25 Jan 2010https://doi.org/10.1161/HYPERTENSIONAHA.109.134205Hypertension. 2010;55:579–592Other version(s) of this articleYou are viewing the most recent version of this article. Previous versions: January 25, 2010: Previous Version 1 Arterial biomineralization processes have been afflicting humans for ≥5 millennia, as realized in 2003 via the computed tomographic imaging of Ötzi, the intriguing "ice mummy" discovered in the Tyrolean Alps.1 Patchy abdominal atherosclerotic calcification was readily detected in the postmortem of this ≈40-year-old hunter of the early Copper Age, by 2000 years a predecessor of King Tutankhamen.1 Today, an epidemic of vascular calcification is emerging within our aging and dysmetabolic populace.2,3 Although vascular calcification was once considered only a passive process of dead and dying cells, work from laboratories worldwide has now highlighted that arterial biomineralization is an actively regulated form of calcified tissue metabolism.4,5 Moreover, as in skeletal development – where unique biology controls matrix mineralization in membranous bone, endochondral bone, dentin, and enamel,6,7 mechanistic diversity exists in the pathobiology of vascular calcium deposition.2,4,5,8 Five common forms of vascular calcification, each possessing unique histoanatomic characteristics and clinical settings with overlapping yet distinct molecular mechanisms, have been described to date4,5,9 (Table 1). Although we touch on the subject, the reader is referred to other contemporary reviews for in-depth consideration of pathogenic differences.2,4,5Table 1. Common Histoanatomic Forms of Vascular Calcification and Clinical Settings2,4,5,11,99TypeCharacteristicsHistopathologyDisease BiologyRisk FactorsCalcific aortic valve disease, also known as calcific aortic stenosis or aortic valve calcificationCalcification of aortic valve leafletsIntracellular lipid and extracellular lipoprotein accumulation in valvesMixed picture of membranous, endochondral, and dystrophic calcificationHypercholesterolemia• Low-density lipoprotein cholesterol• Advanced ageRunx2/Cbfa1, Msx2, and Sox9 osteogenic transcription factors expressed in VICsStenosis with variable regurgitationSubendothelial thickening• β-Catenin osteogenic programs (Wnt3a)135Increased myocardial workloadDisplaced, split elastic lamina• Promoted by oxidative stressLeft ventricular hypertrophy, heart failure, syncope, and sudden deathFibrofatty expansion between fibrosa and ventricularis• Promoted by matrix stiffness144Bicuspid aortic valveMultiple early calcification foci at base of lesions (regions of highest mechanical stress) with later coalescenceNotch and NO synthase 3 genetics contribute in bicuspid disease170Woven bone with marrow in ≈13% of specimens169Cell-mediated inflammatory cells observed in bicuspid and senile calcific diseaseT2DM46 Metabolic syndrome45 Hypertension154 Tobacco use Male sexAmorphous nodules of calcium phosphate accrue via epitaxial mineral depositionAMC, also known as medial artery calcification, Mönckeberg sclerosis, or medial calcific sclerosisCalcification of the arterial tunica mediaCircumferential, contiguous, and confluent calcification of tunica mediaArterial activation of "membranous" ossificationT2DM63–65Reduced vascular compliance and impaired Windkessel physiologyCircumferential adventitial inflammation with fibrofatty expansion Msx2 and Osx earlyAdvanced ageIncreased lower extremity amputation risk in T2DMAKP2-positive matrix vesicles mediate calcification, associated with elastin lamellae Runx2 lateCKD (see below)Increased pulse pressureUsually spares coronary arteries except in setting of CKD and calcium-based phosphate binders24Adventitial-medial Wnt signaling direct osteogenic differentiation of CVCs in tunica mediaAutonomic neuropathy171,172Increased myocardial workload Paracrine Wnt signals from adventitial myofibroblastsMyocardial infarction and stroke β-Catenin osteogenic programs activated in mediaElevated lipidaceous markers of oxidative stressTNF and BMP2 dependent, osteogenic signals propagated by hydrogen peroxide10Autonomic neuropathy171,172Low-grade systemic inflammationAtherosclerotic intimal calcificationCalcification of atherosclerotic plaquesEccentric, lumen-deforming, type Vb intimal plaque173Mostly endochondral ossification picture in addition to lipid core mineralization and fibrous microcalcificationHypercholesterolemia4(Continued)Table 1. ContinuedTypeCharacteristicsHistopathologyDisease BiologyRisk FactorsAnginaSome calcification present in most atherosclerotic plaquesFocal inflammation Low-density lipoprotein cholesterolAcute coronary syndromePatchy distributionOxidized low-density lipoprotein in intima and macrophage-derived signal promote osteogenic differentiation of mural CVCsHypertension154Myocardial infarction, stroke, and sudden deathSeveral mechanismsRunx2/Cbfa1, Sox9>>Msx2 expressionType 1 diabetes mellitusReduced vascular compliance Lipid core calcificationRANKL, TNF, and IL6 dependentT2DMPeripheral arterial disease and claudication Fibrous calcification/apoptotic bodiesMacrophage and T-cell sources of RANKL28,127Tobacco useMitral and aortic annulus calcification are both associated with atherosclerotic intimal calcification172 Endochondral ossification with matrix vesicles, AKP2 positive ElastinolysisRheumatoid arthritis174 Systemic lupus erythematosus175,176Vascular calcification of end-stage kidney disease CKD5CKD5 (GFR <15 cc/min/1.73 m2) in concert with any of the aboveAll the above11,97Impaired serum calcium phosphate homeostasisAny of the above11Impaired serum calcium phosphate homeostasisVSMC apoptosis158Vascular smooth muscle cells elaborate mineralizing matrix vesicles and apoptotic bodies, stimulated by elevated serum calcium and phosphateHyperphosphatemiaOften represents acceleration of antecedent calcific vasculopathyElastinolysis (cathepsin S)Low-grade systemic inflammation, reduced serum fetuin impairs matrix vesicle clearance by VSMCsHypercalcemia 40% diabetic At any level of CKD, patients with diabetes mellitus have greater vascular calcium loads Can and does occur without antecedent disease (eg, pediatric populations)AMC predominates with T2DM as cause of CKD Coronary artery medial calcification can be seen in this setting of CKD24 Frequent mitral annular calcification observedDialysis induces VSMC apoptosis Phosphate upregulates Runx2/Cbfa1 and Msx2 expression in VSMCs via Pit1116,177 Vascular TNF, BMP2, and Msx2 increased, coregulated in CKD90Renal osteodystrophy, low turnover bone disease178174Excessive use of calcium-based phosphate binders and excessive PTH suppression178,179Calcific uremic arteriolopathy, also known as calciphylaxisGFR > acuteArteriolar ( LDLR−/−> wild-type C57Bl/6 mice.53,188,189Diet-induced hypercholesterolemiaYes33,183Yes33,184Diet-induced atherosclerosisYes183Yes185Diet-induced diabetes mellitus with hypertriglyceridemiaNo33Yes33,44Diet-induced obesityLess so33Yes33"Spontaneous" arterial chondroid metaplasiaYes (accelerated by drug-induced diabetes mellitus)54,98No29,44Early diet-induced nonendochondral7 medial artery calcificationNo54Yes29,167Late diet-induced endochondral7 atherosclerotic calcificationYes54Yes28,29,167Hemodynamically significant calcific aortic valve disease occurs with progressionNot known (valve thickening seen with CRI)186Yes (concomitant ApoB100/100 genotype)125,187Exaggerated inflammatory response and susceptibility to mortality with gram-negative sepsisYes53,188Less so53,189*Arterial calcification accelerated by chronic renal insufficiencyYes140Yes118Inflammatory Cytokines in the Initiation and Progression of Arterial Calcification: Lessons Learned From LDLR−/− and ApoE−/− MiceSome degree of vascular inflammation is a frequent concomitant of most forms of arterial calcification.13,14 Sites of inflammation relevant to disease biology may not only include the atherosclerotic intima and media but also the tunica adventitia.15–18 Of note, calcification of the elastic lamina with elastinolysis in the absence of overt histological inflammation has been reported,19–23 and intimal CD68+ macrophage accumulation is more commonly associated with atherosclerotic versus medial calcification.24 However, because calcium phosphate mineral deposition itself elicits inflammatory responses,25 including tumor necrosis factor (TNF) production by macrophages,26,27 a primary role for inflammation in the pathogenesis of clinically relevant vascular calcification was unproven until very recently.28–32 In this section, we review this new data and also highlight distinctions between the LDLR−/− and apoE−/− murine disease models33 (Table 2) that provide insights into the mechanistic complexities of inflammation-dependent arterial calcium accumulation.RANKL and Atherosclerotic CalcificationReceptor Activator of Nuclear Factor κB Ligand/Osteoprotegerin Signaling and Atherosclerotic CalcificationThe first robust evidence for the primary contributions of inflammatory cytokine signaling to pathogenesis of vascular calcification arose from the generation and evaluation of the osteoprotegerin (OPG)−/− mouse.34 OPG-deficient mice develop severe medial and intimal arterial calcification in conjunction with high-turnover osteoporosis driven by excessive osteoclast formation.34 OPG was first shown to function as an antagonistic "faux receptor" of receptor activator of nuclear factor κB ligand (RANKL), the TNF superfamily member that signals via its receptor activator of nuclear factor κB on monocyte/macrophage progenitors to promote the formation of bone-resorbing osteoclasts.7,35 In bone, the antagonist OPG is expressed alongside RANKL in the osteoblast lineage, However, OPG is also expressed in vascular smooth muscle cells and endothelial cells of large arteries, a venue where RANKL is normally absent but induced with inflammation.35 RANKL expression is readily detected in T cells and macrophages near atherosclerotic lesions and within cytokine-stimulated endothelium.35 Intriguingly, RANKL has been shown recently to promote osteochondrogenic mineralization of vascular smooth muscle cells (VSMCs)36 and aortic valve interstitial cells37 in vitro. Via the receptor activator of nuclear factor κB expressed in VSMCs, RANKL upregulates bone morphogenetic protein 4 (BMP4) expression, thus providing an autocrine stimulus for osteogenic differentiation (see also the "Of BMPs and Wnts" section below).36 These dual and disparate actions of RANKL on the skeletal monocyte/macrophage lineage versus VSMCs likely explain the intriguing phenotype of OPG-null mice.34 Of note, although the vascular calcification of OPG deficiency occurs in the complete absence of atheroma formation,34 calcified lesions begin to form in arteries only in the postpartum period with copious CD3+ T-cell infiltrates, a few F4/80+ macrophages, and cathepsin K+ osteoclast-like cells.34,38 This suggests that, in vivo, inflammatory signals absent in utero are necessary for vascular disease initiation and progression in OPG−/− animals. In addition, as first observed in the diabetic LDLR−/− mouse,39 serum levels of OPG are higher in patients with diabetes mellitus.40,41 Because OPG is expressed in VSMCs,42 such increases in the setting of type 2 diabetes mellitus (T2DM) presumably reflect a vascular defense that helps prevents excessive RANKL signaling via negative feedback regulation.28Perturbations in RANKL/OPG Signaling and the Pathobiology of ArteriosclerosisAlthough compelling, the "spontaneous" vascular calcification observed in response to the genetic lesioning in OPG-deficient mice did not ensure contributions to the pathobiology of arteriosclerosis34; however, this caveat has been addressed recently.28 Inhibition of RANKL via administration of recombinant OPG has been evaluated in 2 very different murine models of vascular disease33: the LDLR−/− mouse28 and the apoE−/− mouse.43 It is important to highlight that, whereas both models encompass impaired cholesterol metabolism and atherosis on the C57Bl/6 background, the arteriosclerotic disease processes exhibited by these 2 preclinical models are very distinct (Table 2).33 As Schreyer et al33 first showed, although both models develop atheroma in response to cholesterol-containing fatty diets, the apoE-null mouse never develops the clinically relevant contributions of insulin-resistant diabetes mellitus and obesity with hypertriglyceridemia. However, the male LDLR−/− mouse develops both of these relevant characteristics alongside arterial calcification in response to challenge with fatty diet-possessing compositions typical of westernized societies,33,44 a clinically important stimulus for vascular disease.45,46 Early medial artery calcification is followed by progressively severe atherosclerotic disease in this model (see below).29 Furthermore, the diet-induced systemic low-grade inflammation, characterized by low but measurable levels of circulating TNF in obese LDLR−/− mice29,47 and diabetic humans,48–50 is not seen and apparently does not contribute to vascular inflammation in the apoE−/− model43,51 even when streptozotocin is administered to induce diabetes mellitus.52 However, in response to other stimuli, such as lipopolysaccharide administration or Klebsiella infection, apoE−/− mice exhibit exaggerated TNF induction and increased mortality.53 Finally, in the apoE-null mouse, vascular calcification quickly evolves on the backdrop of VSMC chondroid metaplasia8 that is observed over time even on mouse chow, that is, in the absence of cholesterol-rich dietary challenge.54 By comparison, evolution of arterial calcification in the LDLR−/− mouse is more protracted and elicited by the clinically relevant Western diet (42.00% of calories from fat and 0.15% cholesterol), accruing vascular mineral deposition via sequentially distinct mechanisms.28,29 At early stages, vascular calcification can be histologically detected by Alizarin red staining within the tunica media of major conduit arteries of diabetic, male LDLR−/− mice, biochemically quantifiable after acid extraction.29 Atheromata are not uniformly present at this early stage and, if present, do not stain for calcium. As with atherosclerosis, the initial calcium deposition within the tunica media may be elastin-organized phospholipid vesicles,55,56 because every little inorganic phosphate staining is evident by von Kossa at this stage.29 Similar observations have been described in human specimens.57 With progression, however, massive aortic sinus and subintimal cholesterol deposits accrue, with atherosclerotic calcification visualized within the cholesterol clefts and degenerating atheromata.29 During this second phase, chondroid metaplasia clearly contributes to vascular calcium accrual in male LDLR−/− mice,28 as observed in apoE−/− mice.8 The extent of medial calcium is, thus, increased on Alizarin red staining,29,57 and the von Kossa method for visualizing inorganic phosphate now reveals massive medial and atherosclerotic calcium phosphate deposition in male LDLR−/− mice fed fatty diets.28,29 Thus, when place on high-fat westernized diets, the male LDLR−/− mouse sequentially elaborates an early arterial medial calcification program (Table 1) that, with disease progression, is augmented by processes of atherosclerotic intimal calcification (Table 1; see also Table 2).Inhibition of RANKL Signaling as a Therapeutic Approach to Arteriosclerotic CalcificationAs noted above, OPG is an endogenous inhibitor of RANKL signaling that limits arterial calcium accumulation during development. Recently, the impact of pharmacological inhibition of RANKL by OPG has been evaluated in the above preclinical models of atherosclerosis and arterial calcification. Interestingly, very distinct responses are observed with OPG administration in LDLR−/− and apoE−/− mice.28,43 Morony et al28 first evaluated the male LDLR−/− mouse, the dynamics of endogenous RANKL/OPG signaling during disease initiation and progression, and the impact of exogenous OPG administration. Serum RANKL measurements demonstrated that progression of vascular disease over 5 months of dietary cholesterol challenge closely tracks the progressive recovery of circulating RANKL after an early phase of diet-induced suppression.28 Early diet-induced increases in OPG, a presumed adaptive mechanism to protect against untoward RANKL signaling,36 exhibited no dynamic change with progression.28 As predicted from studies of OPG−/− mice,34 male LDLR−/− mice treated with exogenous OPG exhibit reduced arterial calcification and diminished aortic osteochondrogenic differentiation.28 However, no change in atherosis (ie, the size of arterial atheroma) was observed.28 Intriguingly, 3 sources of vascular RANKL production were identified in this LDLR−/− model: (1) the F4/80+ monocyte-macrophage population in closest proximity to lesions undergoing chondroid metaplasia; (2) the endothelial cells overlying atheroma; and (3) the CD3+ T cells at the adventitial-medial junction.28 Whether any one source of RANKL production represents the lynchpin for the OPG-dependent inhibition of progressive vascular mineral accrual in this model remains to be determined.In apoE−/− mice, as in LDLR−/− mice, OPG administration apparently does not affect atheroma lesion size.43 However, OPG significantly increases fibrous cap size and thickness and reduces matrix metalloproteinase (MMP) 12 levels, potentially stabilizing the lesion but not directly assessed43 (see below). Nonsignificant, tantalizing trends for reductions in numbers of macrophages and T cells were also observed in response to OPG administration. Unlike male LDLR−/− mice, where diet-induced obesity increases circulating TNF levels,29 basal TNF levels are below the limits of detection in apoE−/− animals and, thus, not measurably changed by OPG administration.43 Unfortunately, calcification was not scored in this recent study.43 However, Bennett et al31 have applied murine genetics to carefully detail the important role for endogenous OPG in the calcification of advanced atherosclerotic lesions of apoE−/− mice by generating and evaluating OPG−/−;apoE−/− mice. In this model, congenitally deficient OPG−/−;apoE−/− mice exhibit atherosclerotic lesions of increased size in the innominate artery, with significantly increased areas of calcification and aortic calcium accumulation measured during disease progression.31 Plaque stability was not assessed in OPG−/−;apoE−/− mice, but OPG was shown to increase MMP9 activity in vitro,31 and MMP9 promotes intraplaque hemorrhage in vivo in advanced atherosclerotic lesions of apoE-null animals.58,59 However, congenitally deficient MMP9−/−;apoE−/− mice exhibit increased lesion size after disease initiation versus MMP9-replete siblings,60 suggesting that stage-specific roles of MMP9 exist in atherosis and sclerosis.58 As a modulator of MMP9, OPG could potentially exert adverse, as well as beneficial, arteriosclerotic actions during pharmacological manipulation of RANKL signaling.31 Thus, as in the LDLR−/− mouse, OPG limits arterial calcium accumulation in the apoE-null mouse. OPG may regulate plaque stability, but the differential responses of pharmacological versus genetic manipulation of OPG on vascular histopathology in apoE−/− mice highlight the need for a more detailed assessment of impact on plaque formation, stability, and regression.In summary, antagonism of RANKL signaling cascades holds much promise for modulation of atherosclerotic calcification.61 Of note, a humanized antibody that antagonizes human RANKL has been developed for prevention of fractures in osteoporosis62; based on preclinical studies of Helas et al32 using a "humanized RANKL" murine model, this same reagent might be useful in treatment of cardiovascular calcification. However, the net impact on vascular physiology (vascular compliance, Windkessel-dependent conduit function, distal tissue perfusion, arterial remodeling, and plaque stability) has yet to be determined.Medial Artery Calcification and Diabetic ArteriosclerosisMedial Artery Calcification, Arteriosclerosis, and Lower Extremity Amputation Risk in T2DMThe relationship between arteriosclerotic medial artery calcification (AMC; Table 1) and the risk of lower extremity amputation in T2DM has been appreciated for 2 decades.63,64 The earliest studies were reported for Pima Indians, a native American population with increased risk for T2DM.63,64 Subsequent studies from Finland identified that radiographic femoral medial artery calcification (not atherosclerotic calcification) was the single best predictor of lower extremity amputation in T2DM.65 Why, then, does increased arterial stiffness (arteriosclerosis) in T2DM, arising from AMC without peripheral atherosclerosis, contribute to the increased risk for lower extremity amputation?66 Conduit vessel stiffening from any cause67 compromises normal arterial Windkessel physiology,67,68 thus impairing uniform distal tissue perfusion throughout the cardiac cycle.69,70At this point, however, it should be re-emphasized that critical limb ischemia arising from atherosclerotic plaque formation and arterial stenosis in the femoropopliteal bed is a well-recognized contributor to lower extremity amputation risk; moreover, atherosclerotic calcification also contributes to conduit vessel stiffness.71–74 Medical strategies, such as statins that reduce atherosclerotic disease burden, also improve outcomes in patients with peripheral arterial disease.71,75 Reductions in ankle-brachial indices (ABIs) provide a clinically useful tool for identifying symptomatic individuals at risk.72,73 Increased mobility, reduced claudication, limb salvage, and improved ABIs can often be achieved by surgical or percutaneous vascular interventions,71 more successfully so in stenosed distal femoropopliteal segments76,77 than proximal segments78 and less successfully so in patients with diabetes mellitus.79–81 However, in the setting of T2DM, peripheral arterial disease arises with contributions from both medial artery calcification and atherosclerosis.74 Furthermore, in T2DM, ABIs are frequently elevated,82 not reduced, because of medial calcific sclerosis.74,82 Although elevated ABIs do not necessarily convey increased risk for atherosclerotic disease,83 an ABI ≥1.3 does indicate the presence of arteriosclerosis (ie, arterial stiffening) and concomitantly portends lower extremity amputation.84 In summary, the clinical evaluation of peripheral arterial disease in patients with T2DM requires special consideration, including assessment of toe-brachial indices in lieu of ABIs.82Mechanisms of Medial Artery Calcification in T2DM: Clues From the Field of Bone Biology and the LDLR−/− MouseDuring skeletal mineralization, bone formation can occur via either endochondral (preceding cartilage template required) or membranous (nonendochondral; no cartilage required) processes.7 Osteo/chondrocytic transcription factors, such as Sox9, osteoblast transcription factor runt related transcription factor (Runx) 2, osteoblast transcription factor muscle segment homeobox homolog (Msx) 2, Msx1, and osteoblast transcription factor osterix (Osx), play critical roles in promoting either endochondral (Sox9, Runx2, and Osx) or membranous (Msx2, Msx1, Runx2, and Osx) bone formation.7 In bone, polypeptide morphogens, such at bone morphogenetic proteins (BMPs) and wingless/mouse mammary tumor virus integration site family (Wnts) induce these osteoblast DNA binding proteins along with β-catenin, a transcription coadapter indispensable for bone formation.7,85 A common feature of active osteogenic mineralization is induction of alkaline phosphatase (AKP) 2, the "bone" alkaline phosphatase that degrades the plentiful and endogenous mineralization inhibitor inorganic pyrophosphate (PPi; Figure).7 Of note, Sox9, Runx2, Msx2, and AKP2 have all been described as being expressed in calcifying human arterial segments86 and are upregulated by stimuli that promote arterial calcification (Figure). Download figureDownload PowerPointFigure. Inflammation and osteogenic regulation of vascular calcification: a review and working model. Osteochondrocytic cells that promote vascular matrix mineralization can arise from at least 2 sources: transdifferentiation of VSMCs (ie, a type of phenotypic modulation in which the mature VSMC phenotype is replaced, and reprogrammed to that of an osteochondrocytic cell) or osteogenic lineage allocation from a multipotent mesenchymal progenitor (ie, a cell that has the potential to become an osteoblast, chondrocyte, VSMC, or adipocyte). Both processes are triggered by key inflammatory cytokines and oxidative stress signaling (boxed). VSMCs also elaborate apoptotic bodies and matrix vesicles that can nucleate mineral deposition but also may play a role in removing vascular calciprotein particles via fetuin and MGP-dependent cellular uptake. Thus, apoptosis of VSMCs not only provides substrate for nucleation but also loss of cellular defenses. Multiple paracrine inhibitors control pro-osteogenic signals provided by BMP/Wnt signaling, RANKL, and TNF actions and nucleation/aggregation/epitaxial propagation of apatitic calcium phosphate deposition. Via heat shock protein 70 (HSP70)-mediated inhibition of MGP and AKP2-mediated PPi degradation, inflammatory cytokines, such as IL6 and TNF, impair MGP and PPi defense mechanisms, respectively. Inflammation also downregulates expression of serum fetuin, an import hepatocyte-derived inhibitor of soft tissue mineral deposition. Not shown are the enzymatic defense mechanisms, such as catalase and glutathione peroxidase, that reduce vascular oxidative stress.10,88,168 Although clearly an important stimulus for vascular BMP2 expression,131 remarkably few studies have examined the molecular mechanisms whereby hypertension activates vascular osteogenic signaling cascades. Of note, contribution of marrow-derived osteogenic endothelial progenitor cells as an additional source of mineralizing vascular mesenchymal progenitors has been posited recently but has yet to be established.152 See text for details and additional references.The molecular mechanisms controlling initiation and progression of medial artery calcification in T2DM have been studied recently in detail in the male LDLR−/− mouse (Table 2), a model in which obesity, diabetes mellitus, and osteogenic arterial calcification programs are induced in response to high-fat diets possessing compositions characteristic of westernized societies.2,29,39,44,87 Importantly, diet-induced disease in male LDLR−/− mice2,29,44,87 closely tracks molecular and physiological characteristics of T2DM patients afflicted with valve88,89 and arterial86,90 calcification. A critical clue to the pathogen