点击显示 收起
【摘要】
Objective— Aortic calcification is prevalent in type II diabetes (T2DM), enhancing morbidity and tracking metabolic syndrome parameters. Ldlr –/– 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 –/– 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 β-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.
Type II diabetes (T2DM) promotes medial artery calcification, a significant risk factor for lower extremity amputation. Using a murine disease model—the Ldlr –/– mouse fed high fat diabetogenic diets—we identified that arterial TNF-alpha signaling activates osteogenic Msx2-Wnt gene expression programs that direct medial calcification during disease initiation.
【关键词】 aortic calcification Wnt TNF metabolic syndrome diabetes
Introduction
Type II diabetes (T2DM) increasingly afflicts our dysmetabolic population, with concomitant increases in vascular disease burden. 1 Public health consequences of diabetic macrovascular disease are difficult to overestimate. Stroke, myocardial infarction, congestive heart failure, and lower- extremity amputation exact serious morbidity and economic cost. 1 Expenses associated with amputation are equivalent to the combined yearly costs of managing congestive heart failure and fatal and nonfatal myocardial infarction. 1 Lehto identified aortofemoral medial artery calcification as the most significant predictor of lower-extremity amputation. 2 A better understanding of mechanisms that control aortic calcium deposition will lead to novel strategies for improving macrovascular Windkessel function and thus disease burden.
Arterial calcification is a highly regulated form of tissue mineralization that proceeds via mechanisms resembling membranous and endochondral bone formation. 3 Osteo/chondrogenic transcription factors such as Sox9, Runx2, and Msx2 are upregulated in the mineralizing vasculature, activating expression of osteogenic enzymes and matrix proteins necessary for calcium deposition. 4 Both osteogenic and chondrogenic programs are elaborated in calcifying arterial specimens, including those from diabetic patients. 4 Rajamannan first demonstrated expression of Wnt3 and osteogenic Wnt signaling in calcifying human aortic valves. 5 The Multiethnic Study of Atherosclerosis established stepwise relationships between aortic valve calcium load and metabolic syndrome parameters. 6 Msx2-Wnt signaling participates in the aortic valve and medial calcification characteristic of T2DM in Ldlr –/– mice fed high-fat diets (HFD). 7,8 Thus, the obesity, hyperinsulinemia, hyperglycemia, dyslipidemia, and aortic calcification induced in the Ldlr –/– mouse fed HFD recapitulates key biological features of human disease. 5,6 Mechanisms that activate the aortic Msx2-Wnt cascade in this model have yet to be elucidated.
An emerging view of T2DM encompasses obesity-dependent low-grade systemic inflammation as a component of insulin resistance and vascular disease. 9,10 Adipose is an endocrine tissue that elaborates multiple adipocytokines, including TNF-. 10 Paracrine interactions between tissue adipocytes and interstitial macrophages control production of fat-derived humoral signals. 11 Chief among the inflammatory mediators is TNF-, a prototypic cytokine of activated macrophages 11 that induces insulin resistance. 9,10 TNF- activity of the fibro-fatty arterial adventitial layer is upregulated with diabetes. 12 Demer first identified that TNF- may participate in vascular calcification, upregulating ALP (alkaline phosphate) activity as a necessary component of CVC (calcifying vascular cell) mineralization in vitro. 13 However, contributions of TNF- to aortic calcification have not been studied in vivo.
In this work, we evaluated whether TNF- regulates the osteogenic signals recently identified as participating in aortic calcification in diabetic Ldlr –/– mice. 7,5 We identify that procalcific aortic Msx2-Wnt signaling cascades are entrained to TNF- signals activated by HFD-induced obesity and T2DM.
Materials and Methods
Materials, Cells, and Fluorescence RT-PCR
Recombinant TNF- and Dkk1 were obtained from R&D Systems. ELISA kits for haptoglobin (Life Diagnostics Inc), and TNF- and leptin (RND Systems) were commercially obtained. Biochemical assays for cholesterol and glucose were performed using commercial assays as per the manufacturer (Thermo). The TNF- neutralizing antibody infliximab 14 was purchased from Centocor. Other reagents were purchased from Sigma or Fisher. Primary mouse aortic adventitial myofibroblasts were generated as described 7 (detailed in the supplemental materials, available online at http://atvb.ahajournals. org). Fluorescence RT-PCR was used to quantify the relative mRNA accumulation of aortic osteogenic signaling molecules as previously detailed 7 (detailed in the online supplement).
Generation of SM22-TNFalphaTg and SM22-TNFalphaTg;TOPGAL Transgenic Mice
SM22-TNFalpha transgenic mice were generated as before 7 at the Washington University Mouse Genetics Core. Detailed methods are presented in the accompanying online supplement. TOPGAL mice were purchased from Jackson Laboratory, Bar Harbor, Maine.
Murine Studies and Cardiovascular Histochemistry
Male Ldlr –/– mice (Jackson Labs #002207; C57BL/6J background) were fed Picolab Rodent Diet 20 (#5053) until challenged at 2 months of age with HFD (Harlan Teklad diet TD88137; 42% fat calories, 0.15% cholesterol) for 5 weeks. All studies had 5 to 15 animals per treatment arm as indicated. Animals were dosed twice weekly with 10 µg/g subcutaneous infliximab in vehicle, 15 or with vehicle (500 mg sucrose, 0.5 mg polysorbate 80, 2.2 mg monobasic sodium phosphate monohydrate, and 6.1 mg dibasic sodium phosphate dihydrate in 10 mL of sterile water as per the package insert). At the end of the treatment period, mice were fasted overnight, then euthanized following protocols approved by the institutional Animal Studies Committee. Aortic segments from the arch to the renal arteries were dissected and individually analyzed for either (1) gene expression by RT-PCR or (2) calcium content after formic acid extraction. These published methods 7 are detailed in the accompanying online supplement.
Statistics
For histological staining results, chi-square analysis was performed with Yate correction 16 to compare SM22-TNFalphaTg;TOPGAL versus TOPGAL littermates. For all other data, analyses were performed using Student t test, with data presented as the mean±SEM.
Results
High-Fat Diets Induce TNF-, Low-Grade Systemic Inflammation, and Medial Artery Calcification in Ldlr –/– Mice
The male Ldlr –/– mice fed HFD typical of Western societies develop hyperglycemia, hyperinsulinemia, dyslipidemia, and obesity. 17 As observed in humans with metabolic syndrome or T2DM, 6 aortic calcification is enhanced in male Ldlr –/– with HFD—with the concomitant induction of osteogenic gene regulatory programs in aortic tissues. 8 Alizarin red staining for calcium deposition reveals that, after 1 month of HFD, patchy calcium deposition is observed in the aortic tunica media ( Figure 1A and 1 B). After 6 months of HFD, both medial and atherosclerotic calcium deposition are visible in proximal and distal thoracic aortic segments, with greater staining observed in the tunica media ( Figure 1C and 1 D). As observed in the coronary arteries ( Figure 1 E), regions of aortic media lacking overlying atheroma stained with Alizarin red with an intensity approximating that of mineralizing neonatal mouse bone ( Figure 1 F).
Figure 1. Diabetogenic diets promote calcification in male Ldlr –/– mice. Mice were fed HFD for 1 (A and B) or 6 (C through F) months. Alizarin staining of aorta histologically localized calcium deposition. 7 Initial patchy calcification (A and B) progressively becomes circumferential (C and D). Medial calcification predominates (black arrows, E and F), but some atherosclerotic calcification occurs (white arrows, D, F). A skeletal control for calcium staining is included in panel F.
Prior studies showed that TNF- plays a critical role in CVC mineralization in vitro. 13 Because TNF- signaling is implicated in pathogenesis of T2DM, 9 we evaluated serum TNF- levels in Ldlr –/– mice. Under basal conditions, fasting TNF- levels were undetectable; however, serum TNF- rose to 8.4±3.6 µg/L in animals fed HFD ( P =0.03). Moreover, HFD feeding significantly induced serum haptoglobin levels—a marker of inflammatory cytokine signaling—from 0.46±0.03 µmol/L to 1.27±0.16 ( P =0.025). To identify whether endogenous TNF- contributed to diet-induced inflammatory responses in Ldlr –/– mice, we tested the effects of infliximab (INX)—a clinically useful TNF- –neutralizing antibody—implementing a validated dosing regimen. 14 Circulating levels of haptoglobin were significantly reduced by infliximab, to 0.93±0.12 µmol/L ( P =0.046); parallel reductions in another serum inflammation marker, hemopexin, were also observed (not shown). Infliximab did not reduce the weight gain, hyperglycemia, hypercholesterolemia, or hyperleptinemia induced by HFD ( Table ). Thus, TNF- is upregulated in Ldlr –/– mice fed HFD. Infliximab administration reduced inflammation—reflected in the serum inflammatory biomarker haptoglobin—with little effect on weight gain, hyperglycemia, hypercholesterolemia, and hyperleptinemia induced by HFD consumption.
Table. Metabolic Parameters After 5 Weeks of Dietary Challenge and Infliximab Treatment *
Aortic Osteogenic BMP2-Msx2-Wnt Programs Induced by HFD Are Inhibited by Infliximab, With Concomitant Reduction in Aortic Calcium Content
In response to HFD, Ldlr –/– mice accrue arterial calcium accumulation as revealed by Alizarin red, most notable in the tunica media of the aorta and the coronary arteries ( Figure 1 ). We previously demonstrated that HFD upregulated both aortic BMP2 and Msx2 in Ldlr –/– mice. 8 Of note, paracrine Wnt signals activated by aortic Msx2 expression contribute to medial calcium accrual, 7 and a related paracrine Wnt signaling cascade controls BMP2-dependent ossification in cultured osteoblasts. 18 Therefore, we wished to assess whether antagonizing TNF- signaling with infliximab would ameliorate procalcific aortic BMP2, Msx2, and Wnt signaling in the Ldlr –/– mouse. As shown in Figure 2 A, HFD upregulated aortic mRNA accumulation for BMP2, Msx2, Wnt3 a, and Wnt7a as described. 7,8 Significantly, aortic expression of these HFD-induced signals was downregulated by infliximab ( Figure 2 A). This was paralleled by infliximab-induced downregulation of the cytokine OPN ( osteopontin ), 19 but not Runx2 or Dkk1 ( Figure 2 B). We next assessed effects of infliximab on aortic calcium accumulation after 5 weeks of HFD, an early disease stage when medial calcification predominates ( Figure 1 A), by quantifying acid-extractable calcium. Ldlr –/– animals were placed on HFD as before, and treated with either vehicle (n=10) or 10 µg/gm infliximab (n=5) twice weekly for 5 weeks. Aortic matrix calcium accumulation was reduced by 30% in animals treated with infliximab ( Figure 2 C, from 1.44 to 0.99 µg calcium/gm aortic tissue; P <0.05). Thus, infliximab, a specific TNF- neutralizing antibody, reduces aortic proosteogenic signaling and early calcium accumulation associated with HFD feeding in male Ldlr –/– mice.
Figure 2. Infliximab downregulates osteogenic BMP2-Msx2-Wnt programs in Ldlr –/– mice fed HFD. A, HFD upregulated aortic BMP2, Msx2, Wnt3a, and Wnt7a. 7,8 Infliximab treatment inhibited induction. B, Infliximab exerted little effect on Runx2 and Dkk1. C, Infliximab reduced aortic calcium in Ldlr –/– mice fed HFD for 5 weeks. At this early stage, only patchy medial calcification occurs ( Figure 1A and 1 B).
TNF- Directly Induces Msx2-Wn t Signaling in Aortic Myofibroblasts In Vitro
Endothelial production of BMP2 is enhanced by TNF-, 20 and BMP2 promotes osteogenic Msx2 8 and Wnt 18 signals. We wished to assess whether TNF- might exert direct actions on aortic Msx2-Wnt signaling. 7 We focused on primary aortic adventitial myofibroblasts, a mural progenitor cell population that expresses Msx2 and generates vascular cells that calcify in vivo and in vitro. 7,8,21 As shown in Figure 3 A, TNF- dose-dependently increased Msx2 mRNA accumulation in aortic adventitial myofibroblasts. Induction was an immediate-early response, because cycloheximide treatment did not prevent Msx2 upregulation (not shown). Subsequent time course studies revealed that Msx2 induction preceded that of BMP2 in myofibroblasts ( Figure 3 B). Moreover, addition of the BMP2 inhibitor, noggin, 18 did not alter TNF- induction of Msx2 (not shown).
Figure 3. TNF- directly activates Msx2-Wnt signaling with subsequent ALP induction. Aortic myofibroblasts 7 were treated with TNF- and analyzed for Msx2 expression. A, TNF- treatment (3 hours) increased Msx2. B, TNF- (10 ng/mL, 2 hours) upregulated Msx2 before BMP2. C, TNF- selectively upregulated Wnt3a and Wnt7a. D, Induction of ALP 7,8 is inhibited by the Wnt antagonist Dkk1. 18
We next evaluated effects of TNF- on expression of Wnt1, Wnt3a, and Wnt7a —canonical Wnt ligands previously identified as components of the aortic Msx2-Wnt signaling cascade. 7 TNF- treatment significantly upregulated expression of Wnt3a and Wnt7a, but had no effect on Wnt1 ( Figure 3 C). TNF- induction of these 2 canonical Wnt ligands was functionally important; recombinant Dkk1—an antagonist of Wnt signaling via LRP5 and LRP6 18 —inhibited TNF- upregulation of bone ALP ( Figure 3 D), a key genomic target of osteogenic Wnt signaling. 7,18 Thus, TNF- can stimulate Msx2-Wnt signaling in aortic adventitial myofibroblasts.
The SM22-TNFalpha Transgene Upregulates Aortic Msx2 Expression and Canonical Wnt Signaling In Vivo
To confirm that TNF- upregulates aortic Msx2-Wnt signaling in vivo, we generated SM22-TNFalpha transgenic (Tg) mice as a model for study. The SM22 promoter has proved useful for directing vascular smooth muscle cell (VSMC)-specific gene expression in vivo. 22 Therefore, we assembled an SM22 promoter— TNF - cDNA expression construct ( Figure 4 A), and generated and characterized SM22-TNFalphaTg mice. SM22-TNFalphaTg mice exhibit 2.4-fold elevated aortic TNF - message by RT-PCR as compared with nontransgenic littermates ( P =0.0003). Moreover, as observed in vitro, the SM22-TNFalpha transgene significantly upregulated expression of aortic BMP2, Msx2, Wnt3a, and Wnt7a gene expression in vivo ( Figure 4 B).
Figure 4. The SM22-TNFalpha transgene augments aortic Msx2-Wnt signaling. A, SM22-TNFalpha expression vector. Transgenic (Tg) mice exhibit 2.4-fold greater aortic TNF - vs nontransgenic littermates ( P =0.0003). B, SM22-TNFalpha Tg mice upregulate aortic BMP2, Msx2, Wnt3a, Wnt7a, and OPN. C and D, β-galactosidase/LacZ histochemistry revealed medial signal in SM22-TNFalphaTg;TOPGAL mice (4/5), not in TOPGAL siblings ( P =0.05, Yate Chi-squared test).
We wished to assess whether the TNF - transgene was capable of augmenting aortic Wnt signaling in vivo; this was important, because TNF- exerts time-dependent biphasic rapid inductive/delayed suppressive effects on myofibroblast Dkk1 expression (unpublished observations). The TOPGAL (TCF/LEF optimal promoter β-galactosidase/LacZ reporter) mouse has been used to monitor activation of canonical Wnt signaling in vivo. 7,23 Therefore, we generated SM22-TNFalphaTg;TOPGAL mice (mixed B6:CD1 background), and compared aortic LacZ staining to that observed with TOPGAL littermates lacking SM22-TNFalpha transgene. Four of 5 SM22-TNFalphaTg:TOPGAL mice exhibited prominent LacZ staining of mural cells ( Figure 4 C, right panels). By contrast, no LacZ staining was observed in 5 of 5 TOPGAL siblings lacking the SM22-TNFalpha transgene ( Figure 4 C, left panels; P =0.05, Yate corrected chi-squared test). Identical results were observed in coronary arteries ( Figure 4 D). Thus, TNF- promotes aortic Msx2, Wnt3a, and Wnt7a expression, and augments aortic canonical Wnt signaling in vivo.
Discussion
The epidemic of diabetes and obesity assailing Westernized societies threatens to interact with the prevalent, age-related incidence of aortic disease to increase macrovascular disease burden. 3 Primary prevention strategies are critically important; however, as many as one-third of patients with T2DM may be unaware of their disorder—and vascular disease processes that threaten life, limb, and autonomy progress from the earliest phases of the dysmetabolic state. 1,2 A better understanding of aortofemoral disease in T2DM is necessary to develop new strategies to address this burgeoning clinical need.
The Ldlr –/– mouse has emerged as one useful model for studying macrovascular injury in response to T2DM. 17 When fed HFD, these mice become obese, with concomitant hyperglycemia and dyslipidemia. 17 Unlike the Apoe –/– mouse—a model of atherosclerosis in the absence of hyperglycemia and obesity 17 Ldlr –/– mice fed the HFD elaborate key features of metabolic syndrome. 17 The relationships between metabolic syndrome parameters and aortic valve calcification in humans were recently established. 6 Thus, the Ldlr –/– murine model faithfully recapitulates key aortic pathobiology, including aortic calcification, entrained to the metabolic syndrome–T2DM continuum of disease severity.
Using this model, we describe a novel TNFalpha– Msx2-Wnt signaling axis that contributes to the pathobiology of diabetic macrovascular disease. TNF- is the prototypic inflammatory cytokine that exerts global influences on metabolism and innate immunity. 9,10 The TNF- neutralizing antibody infliximab downregulated aortic Msx2-Wnt programs without improving fasting serum glucose or cholesterol. Thus, based on the prior studies of Demer 24 and our results, we anticipate that programs elicited by TNF- divert multipotent CVCs—macrovascular mural pericytes 25 —to the osteogenic lineage via activation of the Msx2-Wnt pathway. Of note, in the dyslipidemic Apoe –/– mouse, migratory mesenchymal progenitors are recruited from the adventitia to populate mural VSMC populations. 26 This may include the pericytes, 25 myofibroblasts, 8 and CVCs 24 that program arterial calcification, because surgical resection of the adventitia reduces arterial calcification. 21 Our in vitro studies have emphasized responses in adventitial myofibroblast preparations. Like CVCs generated from tunica media, 24 aortic adventitial myofibroblast preparations are heterogeneous for calcifying cells. 8 As surface markers emerge that permit isolation of VSMCs at each stage of differentiation, it will be important to determine whether TNF- regulation of arterial Msx2-Wnt cascades change with phenotypic maturation.
Proosteogenic signals are counter-balanced by critically important negative regulators of arterial mineral deposition. Chief among these are OPN, inorganic pyrophosphate, fetuin, osteoprotegerin, and matrix Gla protein (MGP). 27,28 Regulatory cross-talk between proosteogenic signals and these mineralization inhibition mechanisms has recently been described. 3 BMP2 upregulates ALP, the ectoenzyme that degrades tissue pyrophosphate—a critical inhibitor of biomineralization and secretagogue for OPN. 19 For full elaboration of the vascular tissue mineralization process, these important defense mechanisms must become compromised with disease progression.
Other metabolic insults—such as the hyperphosphatemia, hypercalcemia, and oxidative stress of uremia 29 —clearly contribute to vascular calcium load via phosphate transporter activation of Cbfa1/Runx2. 30 Runx2 is an osteogenic and chondrogenic transcription factor exquisitely regulated by posttranslation modifications, subnuclear localization, and modulatory protein–protein interactions. 30 With atherosclerotic calcification, endochondral mineralization programs are recruited, primarily dependent on Runx2 and Sox9. 4 Oxysterols generated by lipoprotein oxidation enhance Runx2-dependent mineralization in culture. 31,32 In the male Ldlr –/– mouse fed HFD, Alizarin red staining for calcium accumulation reveals that medial calcification predominates as obesity and T2DM ensue. However, with prolonged dietary challenge of Ldlr –/– mice, atherosclerotic calcification clearly becomes super-imposed in the aorta ( Figure 1 ). Whether the TNF- –dependent Msx2-Wnt pathways and Runx2-regulated programs differentially contribute with disease progression has yet to be explored in this model.
Of note, biomineralization can be nucleated by phospholipid-rich matrix vesicles 33 and apoptotic bodies, 33 or by devitalized elastin. 34 Thus, disease pathobiology and histological characteristics will differ depending on the mechanisms used to initiate vascular calcium deposition. In early lesions of the Ldlr –/– mouse, arterial calcium deposition visualized by Alizarin red staining is overlapping yet distinct from that seen with von Kossa stain for phosphate deposition (unpublished observations); similar observations were noted almost 40 years ago by Puchtler et al in studies of early human atherosclerotic lesions. 35 Moreover, changes in bone resorption—coupled with common, clinically relevant agents such as warfarin that cripple Gla-dependent inhibitors of calcification—can enhance elastin-based arterial calcification by perturbing serum nucleator/nucleation inhibitor homeostasis. 36,37 Indeed, T2DM is frequently accompanied by chronic kidney disease that will increase bone turnover. 29 Thus, a comprehensive understanding of arterial calcification will require integration of the diverse mechanisms that control vascular mineral deposition—and recognition that mechanistic contributions likely change with clinical setting and disease stage. 3
We demonstrate that aortic mineralization programs characteristic of membranous bone formation 38 are entrained to signals elicited by TNF-. TNF- promotes aortic Msx2 expression and enhances procalcific arterial Msx2-Wnt cascades in vitro and in vivo. Treatment of aortic myofibroblasts with Dkk1, an antagonist of paracrine Wnt signals, inhibits TNF- induction of ALP, an early osteogenic gene downstream of Msx2-Wnt activation. Of note, the antiinflammatory compound salicylate also downregulates aortic Msx2 in vivo (not shown). Thus, strategies that attenuate proinflammatory TNF- signals may help ameliorate aortic calcium accumulation.
There are limitations to our study. The magnitude of aortic Msx2 and Wnt message downregulation by infliximab was consistently greater than the observed inhibition of aortic calcium accumulation (30% reduction) at this early disease stage. Regulatory mechanisms in addition to those emphasized in this study must also contribute. These include elastin metabolism, 34,39 fetuin- and calcium phosphate-dependent regulation of matrix vesicle metabolism, 33 osteoprotegerin-inhibited cytokines, 3,36 and other emerging inhibitors of calcification. 25 Terkeltaub 40 recently extended the seminal observations of Giachelli 19 that highlight the importance of vascular OPN; pyrophosphate generating systems not only inhibit mineralization directly, but also stimulate secretion of OPN 40 —a potent inhibitor of vascular calcium deposition and mediator of calcium egress. 19 Of note, infliximab therapy downregulates aortic OPN, consistent with its antiinflammatory properties but potentially compromising beneficial actions of OPN that limit vascular calcium deposition. 19 Moreover, unlike effects on Msx2-Wnt signals, infliximab did not reverse HFD-induced aortic Runx2 expression. With prolonged dietary challenge, endochondral mineralization mechanisms driven by Runx2 may continue unabated, even when Msx2-Wnt signals are mitigated. Finally, key redox enzymes impact the type and extent of oxidative stress—and thus generation of vascular oxysterols from LDL. 31,32 Oxysterols that drive Runx2 activation and endochondral mineralization 31,32 likely participate in aortic calcium load with prolonged dyslipidemia. Future studies will evaluate whether TNF- differentially impacts early versus late calcification mechanisms as disease progresses. Nonetheless, the identification that aortic calcification of T2DM occurs in part via TNF- driven Msx2-Wnt signaling 5 provides insights useful for developing novel multimodality strategies 5,36 to ameliorate diabetic vascular disease. 2,6
Acknowledgments
Sources of Funding
This work was supported by NIH grants HL81138 and HL69229 (to D.A.T.), the Barnes-Jewish Hospital Foundation (to D.A.T.), and St. Louis University (to Z.A.A.).
Disclosures
Dr Towler serves as a consultant for Program Project P01HL030568 and for the Center for Scientific Review Skeletal Biology Development and Disease Study Section.
【参考文献】
Clarke P, Gray A, Legood R, Briggs A, Holman R. The impact of diabetes-related complications on healthcare costs: results from the United Kingdom Prospective Diabetes Study (UKPDS Study No. 65). Diabet Med. 2003; 20: 442–450.
Lehto S, Niskanen L, Suhonen M, Ronnemaa T, Laakso M. Medial artery calcification. A neglected harbinger of cardiovascular complications in non-insulin-dependent diabetes mellitus. Arterioscler Thromb Vasc Biol. 1996; 16: 978–983.
Abedin M, Tintut Y, Demer LL. Vascular calcification: mechanisms and clinical ramifications. Arterioscler Thromb Vasc Biol. 2004; 24: 1161–1170.
Tyson KL, Reynolds JL, McNair R, Zhang Q, Weissberg PL, Shanahan CM. Osteo/chondrocytic transcription factors and their target genes exhibit distinct patterns of expression in human arterial calcification. Arterioscler Thromb Vasc Biol. 2003; 23: 489–494.
Rajamannan NM, Subramaniam M, Caira F, Stock SR, Spelsberg TC. Atorvastatin inhibits hypercholesterolemia-induced calcification in the aortic valves via the Lrp5 receptor pathway. Circulation. 2005; 112: I229–I234.
Katz R, Wong ND, Kronmal R, Takasu J, Shavelle DM, Probstfield JL, Bertoni AG, Budoff MJ, O?Brien KD. Features of the metabolic syndrome and diabetes mellitus as predictors of aortic valve calcification in the Multi-Ethnic Study of Atherosclerosis. Circulation. 2006; 113: 2113–2119.
Shao JS, Cheng SL, Pingsterhaus JM, Charlton-Kachigian N, Loewy AP, Towler DA. Msx2 promotes cardiovascular calcification by activating paracrine Wnt signals. J Clin Invest. 2005; 115: 1210–1220.
Cheng SL, Shao JS, Charlton-Kachigian N, Loewy AP, Towler DA. MSX2 promotes osteogenesis and suppresses adipogenic differentiation of multipotent mesenchymal progenitors. J Biol Chem. 2003; 278: 45969–45977.
Alexandraki K, Piperi C, Kalofoutis C, Singh J, Alaveras A, Kalofoutis A. Inflammatory process in type 2 diabetes: The role of cytokines. Ann N Y Acad Sci. 2006; 1084: 89–117.
Guzik TJ, Mangalat D, Korbut R. Adipocytokines - novel link between inflammation and vascular function? J Physiol Pharmacol. 2006; 57: 505–528.
Lumeng CN, Bodzin JL, Saltiel AR. Obesity induces a phenotypic switch in adipose tissue macrophage polarization. J Clin Invest. 2007; 117: 175–184.
Zhang L, Zalewski A, Liu Y, Mazurek T, Cowan S, Martin JL, Hofmann SM, Vlassara H, Shi Y. Diabetes-induced oxidative stress and low-grade inflammation in porcine coronary arteries. Circulation. 2003; 108: 472–478.
Tintut Y, Patel J, Parhami F, Demer LL. Tumor necrosis factor-alpha promotes in vitro calcification of vascular cells via the cAMP pathway. Circulation. 2000; 102: 2636–2642.
Ross SE, Williams RO, Mason LJ, Mauri C, Marinova-Mutafchieva L, Malfait AM, Maini RN, Feldmann M. Suppression of TNF-alpha expression, inhibition of Th1 activity, and amelioration of collagen-induced arthritis by rolipram. J Immunol. 1997; 159: 6253–6259.
Grounds MD, Davies M, Torrisi J, Shavlakadze T, White J, Hodgetts S. Silencing TNFalpha activity by using Remicade or Enbrel blocks inflammation in whole muscle grafts: an in vivo bioassay to assess the efficacy of anti-cytokine drugs in mice. Cell Tissue Res. 2005; 320: 509–515.
Franceschi D, Crowe J, Zollinger R, Duchesneau R, Shenk R, Stefanek G, Shuck JM. Biopsy of the breast for mammographically detected lesions. Surg Gynecol Obstet. 1990; 171: 449–455.
Schreyer SA, Vick C, Lystig TC, Mystkowski P, LeBoeuf RC. LDL receptor but not apolipoprotein E deficiency increases diet-induced obesity and diabetes in mice. Am J Physiol Endocrinol Metab. 2002; 282: E207–E214.
Rawadi G, Vayssiere B, Dunn F, Baron R, Roman-Roman S. BMP-2 controls alkaline phosphatase expression and osteoblast mineralization by a Wnt autocrine loop. J Bone Miner Res. 2003; 18: 1842–1853.
Steitz SA, Speer MY, McKee MD, Liaw L, Almeida M, Yang H, Giachelli CM. Osteopontin inhibits mineral deposition and promotes regression of ectopic calcification. Am J Pathol. 2002; 161: 2035–2046.
Csiszar A, Smith KE, Koller A, Kaley G, Edwards JG, Ungvari Z. Regulation of bone morphogenetic protein-2 expression in endothelial cells: role of nuclear factor-kappaB activation by tumor necrosis factor-alpha, H2O2, and high intravascular pressure. Circulation. 2005; 111: 2364–2372.
Bujan J, Bellon JM, Sabater C, Jurado F, Garcia-Honduvilla N, Dominguez B, Jorge E. Modifications induced by atherogenic diet in the capacity of the arterial wall in rats to respond to surgical insult. Atherosclerosis. 1996; 122: 141–152.
Akyurek LM, Yang ZY, Aoki K, San H, Nabel GJ, Parmacek MS, Nabel EG. SM22alpha promoter targets gene expression to vascular smooth muscle cells in vitro and in vivo. Mol Med. 2000; 6: 983–991.
DasGupta R, Fuchs E. Multiple roles for activated LEF/TCF transcription complexes during hair follicle development and differentiation. Development. 1999; 126: 4557–4568.
Tintut Y, Alfonso Z, Saini T, Radcliff K, Watson K, Bostrom K, Demer LL. Multilineage potential of cells from the artery wall. Circulation. 2003; 108: 2505–2510.
Collett GD, Sage AP, Kirton JP, Alexander MY, Gilmore AP, Canfield AE. Axl/phosphatidylinositol 3-kinase signaling inhibits mineral deposition by vascular smooth muscle cells. Circ Res. 2007; 100: 502–509.
Hu Y, Zhang Z, Torsney E, Afzal AR, Davison F, Metzler B, Xu Q. Abundant progenitor cells in the adventitia contribute to atherosclerosis of vein grafts in ApoE-deficient mice. J Clin Invest. 2004; 113: 1258–1265.
Reynolds JL, Joannides AJ, Skepper JN, McNair R, Schurgers LJ, Proudfoot D, Jahnen-Dechent W, Weissberg PL, Shanahan CM. Human vascular smooth muscle cells undergo vesicle-mediated calcification in response to changes in extracellular calcium and phosphate concentrations: a potential mechanism for accelerated vascular calcification in ESRD. J Am Soc Nephrol. 2004; 15: 2857–2867.
Zebboudj AF, Imura M, Bostrom K. Matrix GLA protein, a regulatory protein for bone morphogenetic protein-2. J Biol Chem. 2002; 277: 4388–4394.
Moe SM, Chen NX. Pathophysiology of vascular calcification in chronic kidney disease. Circ Res. 2004; 95: 560–567.
Steitz SA, Speer MY, Curinga G, Yang HY, Haynes P, Aebersold R, Schinke T, Karsenty G, Giachelli CM. Smooth muscle cell phenotypic transition associated with calcification: upregulation of Cbfa1 and downregulation of smooth muscle lineage markers. Circ Res. 2001; 89: 1147–1154.
Parhami F, Morrow AD, Balucan J, Leitinger N, Watson AD, Tintut Y, Berliner JA, Demer LL. Lipid oxidation products have opposite effects on calcifying vascular cell and bone cell differentiation. A possible explanation for the paradox of arterial calcification in osteoporotic patients. Arterioscler Thromb Vasc Biol. 1997; 17: 680–687.
Dwyer JR, Sever N, Carlson M, Nelson SF, Beachy PA, Parhami F. Oxysterols are novel activators of the hedgehog signaling pathway in pluripotent mesenchymal cells. J Biol Chem. 2007; 282: 8959–8968.
Reynolds JL, Skepper JN, McNair R, Kasama T, Gupta K, Weissberg PL, Jahnen-Dechent W, Shanahan CM. Multifunctional roles for serum protein fetuin-a in inhibition of human vascular smooth muscle cell calcification. J Am Soc Nephrol. 2005; 16: 2920–2930.
Price PA, Chan WS, Jolson DM, Williamson MK. The elastic lamellae of devitalized arteries calcify when incubated in serum: evidence for a serum calcification factor. Arterioscler Thromb Vasc Biol. 2006; 26: 1079–1085.
Puchtler H, Meloan SN, Terry MS. On the history and mechanism of alizarin and alizarin red S stains for calcium. J Histochem Cytochem. 1969; 17: 110–124.
Price PA, June HH, Buckley JR, Williamson MK. Osteoprotegerin inhibits artery calcification induced by warfarin and by vitamin D. Arterioscler Thromb Vasc Biol. 2001; 21: 1610–1616.
Speer MY, McKee MD, Guldberg RE, Liaw L, Yang HY, Tung E, Karsenty G, Giachelli CM. Inactivation of the osteopontin gene enhances vascular calcification of matrix Gla protein-deficient mice: evidence for osteopontin as an inducible inhibitor of vascular calcification in vivo. J Exp Med. 2002; 196: 1047–1055.
Cohen MM Jr. The new bone biology: pathologic, molecular, and clinical correlates. Am J Med Genet A. 2006; 140: 2646–2706.
Simionescu A, Simionescu DT, Vyavahare NR. Osteogenic responses in fibroblasts activated by elastin degradation products and transforming growth factor-beta1: role of myofibroblasts in vascular calcification. Am J Pathol. 2007; 171: 116–123.
Johnson K, Polewski M, van Etten D, Terkeltaub R. Chondrogenesis mediated by PPi depletion promotes spontaneous aortic calcification in NPP1 –/– mice. Arterioscler Thromb Vasc Biol. 2005; 25: 686–691.
作者单位：Department of Medicine (Z.A.-A.), Division of Nephrology, St. Louis VA Medical Center, St. Louis University, and the Department of Medicine (J.-S.S., C.-F.L., E.H., J.C., A.B., S.-L.,C., D.A.T.), Center for Cardiovascular Research, Division of Bone & Mineral Diseases, Washington University, St.