In the aorta, diabetes activates an osteogenic program that includes expression of bone morphogenetic protein-2 (BMP2) and the osteoblast homeoprotein Msx2. To evaluate BMP2-Msx2 signaling in vascular calcification, we studied primary aortic myofibroblasts. These cells express vascular smooth muscle cell (VSMC) markers, respond to BMP2 by up-regulating Msx2, and undergo osteogenic differentiation with BMP2 treatment or transduction with a virus encoding Msx2. The osteoblast factor osterix (Osx) is up-regulated 10-fold by Msx2, but Runx2 mRNA is unchanged; the early osteoblast marker alkaline phosphatase increases 50-fold with mineralized nodule formation enhanced 30-fold. Adipocyte markers are concomitantly suppressed. To better understand Msx2 actions on osteogenesis versus adipogenesis, mechanistic studies were extended to C3H10T1/2 mesenchymal cells. Msx2 enhances osteogenic differentiation in synergy with BMP2. Osteogenic actions depend upon intrinsic Msx2 DNA binding; the gain-of-function variant Msx2(P148H) directs enhanced mineralization, whereas the binding-deficient variant Msx2(T147A) is inactive. Adipogenesis (lipid accumulation, Pparg expression) is inhibited by Msx2. By contrast, suppression of adipogenesis does not require Msx2 DNA binding; inhibition occurs in part via proteinprotein interactions with C/EBP␣ that control Pparg transcription. Thus, Msx2 regulates osteogenic versus adipogenic differentiation of aortic myofibroblasts. Myofibroblasts capable of both fates can be diverted to the osteogenic lineage by BMP2-Msx2 signaling and contribute to vascular calcification.Mineral deposition in the skeleton is regulated by morphogenetic, metabolic, mechanical, inflammatory, and endocrine factors. With aging, abnormalities in orthotopic (e.g. bone formation) and heterotopic arterial vascular calcification are observed with very high prevalence (1), the latter enhanced by hyperglycemia, hyperlipidemia, and chronic renal insufficiency (1, 2). At least three variants of vascular calcification have been described: (a) calcification of necrotic, intimal atherosclerotic plaques; (b) medial artery calcification; and (c) calcific sclerosis of the aortic valve. Vascular calcification is a highly significant complication of diabetes and has emerged as a powerful predictor of cardiovascular morbidity and mortality (2). The molecular mechanisms that perturb normal vascular calcium metabolism are only beginning to be understood (1, 3, 4). Demer et al. (5) was the first to show that vascular calcification may progress via molecular processes similar to osteogenesis. This group showed that the powerful bone morphogen, bone morphogenetic protein 2 (BMP2) 1 is expressed in calcified atherosclerotic plaques of humans (5). Bostrom et al. (6) further demonstrated that aortic calcification in response to matrix Gla protein deficiency was most likely via BMP2 signaling; matrix Gla protein can abrogate alkaline phosphatase (ALP) induction by inhibiting BMP2 association with the BMP receptor (6). Thus, these studies poi...

No abstract

Objective-Aortic calcification is prevalent in type II diabetes (T2DM), enhancing morbidity and tracking metabolic syndrome parameters. Ldlr Ϫ/Ϫ mice fed high-fat "Westernized" diets (HFD) accumulate aortic calcium primarily in the tunica media, mediated via osteogenic morphogens and transcriptional programs that induce aortic alkaline phosphatase (ALP). Because elevated TNF-␣ is characteristic of obesity with T2DM, we examined contributions of this inflammatory cytokine. Methods and Results-HFD promoted obesity, hyperglycemia, and hyperlipidemia, and upregulated serum TNF-␣ in Ldlr Ϫ/Ϫ mice. Serum haptoglobin (inflammatory marker) was increased along with aortic expression of BMP2, Msx2, Wnt3a, and Wnt7a. Dosing with the TNF-␣ neutralizing antibody infliximab did not reduce obesity, hypercholesterolemia, or hyperglycemia; however, haptoglobin, aortic BMP2, Msx2, Wnt3a, and Wnt7a and aortic calcium accumulation were downregulated by infliximab. Mice with vascular TNF-␣ augmented by a transgene (SM22-TNF␣Tg) driven from the SM22 promoter upregulated aortic Msx2, Wnt3a, and Wnt7a. Furthermore, SM22-TNF␣Tg;TOPGAL mice exhibited greater aortic ␤-galactosidase reporter staining versus TOPGAL sibs, indicating enhanced mural Wnt signaling. In aortic myofibroblast cultures, TNF-␣ upregulated Msx2, Wnt3a, Wnt7a, and ALP. ALP induction was inhibited by Dkk1, an antagonist of paracrine Wnt actions. Conclusions-TNF-␣ promote aortic Msx2-Wnt programs that contribute to aortic calcium accumulation in T2DM.

Abstract-Vascular calcification increasingly afflicts our aging and dysmetabolic population. Once considered a passive process, it has emerged as an actively regulated form of calcified tissue metabolism, resembling the mineralization of endochondral and membranous bone. Executive cell types familiar to bone biologists, osteoblasts, chondrocytes, and osteoclasts, are seen in calcifying macrovascular specimens. Lipidaceous matrix vesicles, with biochemical and ultrastructural "signatures" of skeletal matrix vesicles, nucleate vascular mineralization in diabetes, dyslipidemia, and uremia. Skeletal morphogens (bone morphogenetic protein-2 (BMP) and BMP4 and Wnts) divert aortic mesoangioblasts, mural pericytes (calcifying vascular cells), or valve myofibroblasts to osteogenic fates. Paracrine signals provided by these molecules mimic the epithelialmesenchymal interactions that induce skeletal development. Vascular expression of pro-osteogenic morphogens is entrained to physiological stimuli that promote calcification. Inflammation, shear, oxidative stress, hyperphosphatemia, and elastinolysis provide stimuli that: (1) promote vascular BMP2/4 signaling and matrix remodeling; and (2) compromise vascular defenses that limit calcium deposition, inhibit osteo/chondrogenic trans-differentiation, and enhance matrix vesicle clearance. In this review, we discuss the biology of vascular calcification. We highlight how aortic fibrofatty tissue expansion (adventitia, valve interstitium), the adventitial-medial vasa, vascular matrix, and matrix vesicle metabolism contribute to the regulation of aortic calcium deposition, with greatest emphasis placed on diabetic vascular disease. Key Words: diabetes Ⅲ vascular calcification Ⅲ Wnt signaling Ⅲ bone morphogenetic proteins Ⅲ oxylipids W ith advanced age, vascular inflammation, hypertension, and certain metabolic disorders, calcium accumulates in the arterial macrovasculature. 1 Calcification of aortic valve leaflets and atherosclerotic plaques have long been recognized as clinically important. 2 However, medial artery calcification (MAC) also portends mortality and amputation risk. 1,[3][4][5] Studies of vascular calcified tissue metabolism significantly lag behind those of skeletal metabolism. Executive cell types familiar to bone biologists are seen in calcifying aortic specimens. 1,6 As in bone, endothelial, mesenchymal, and hematopoietic cell lineages control vascular mineral accumulation, with cellular activities entrained to morphogenetic, metabolic, inflammatory, and mechanical demands placed on each vascular segment. 1 We provide a brief overview of vascular calcification, emphasizing how paracrine osteogenic signals recruited by dysmetabolic insults promote aortic calcium deposition in diabetic vascular disease. We point to emerging evidence that inflammation, mechanical, and metabolic oxidative stresses not only provide stimuli that induce vascular osteogenic morphogens but also compromise defense mechanisms that limit vascular calcium deposition. 1 Aortic MACMAC is a high...

A rterial biomineralization processes have been afflicting humans for Ն5 millennia, as realized in 2003 via the computed tomographic imaging of Ö tzi, the intriguing "ice mummy" discovered in the Tyrolean Alps. 1 Patchy abdominal atherosclerotic calcification was readily detected in the postmortem of this Ϸ40-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 date 4,5,9 (Table 1). Although we touch on the subject, the reader is referred to other contemporary reviews for in-depth consideration of pathogenic differences. 2,4,5 In this brief review and perspective, we recount recent data that emphasize inflammation and oxidative stress signaling as key contributors to the pathogenesis of vascular mineral deposition. 10 Furthermore, we highlight differences between the low-density lipoprotein receptor (LDLR)-deficient and apolipoprotein E (apoE)-deficient murine models ( Table 2) that help articulate the multifaceted contributions of dyslipidemia, diabetes mellitus, and uremia to arterial calcium deposition. 2,4,11 We end by summarizing the importance of considering these disease stage-and context-specific contributions arterial mineralization when crafting therapeutic strategies to address the disease burden of vascular calcification that increasingly afflicts our patients. 5 28 -32 In this section, we review this new data and also highlight distinctions between the LDLR Ϫ/Ϫ and apoE Ϫ/Ϫ murine disease models 33 ( Table 2) that provide insights into the mechanistic complexities of inflammation-dependent arterial calcium accumulation. RANKL and Atherosclerotic CalcificationReceptor Activator of Nuclear Factor B Ligand/ Osteoprotegerin Signaling and Atherosclerotic Calcification The 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) Ϫ/Ϫ 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 B ligand (RANKL), the TNF superfamil...