Aortic Msx2-Wnt Calcification Cascade Is Regulated by TNF-α–Dependent Signals in Diabetic Ldlr −/− Mice

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Abstract
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-α 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-α in Ldlr−/− mice. Serum haptoglobin (inflammatory marker) was increased along with aortic expression of BMP2, Msx2, Wnt3a, and Wnt7a. Dosing with the TNF-α 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-α augmented by a transgene (SM22-TNFαTg) driven from the SM22 promoter upregulated aortic Msx2, Wnt3a, and Wnt7a. Furthermore, SM22-TNFαTg;TOPGAL mice exhibited greater aortic β-galactosidase reporter staining versus TOPGAL sibs, indicating enhanced mural Wnt signaling. In aortic myofibroblast cultures, TNF-α upregulated Msx2, Wnt3a, Wnt7a, and ALP. ALP induction was inhibited by Dkk1, an antagonist of paracrine Wnt actions. Conclusions— TNF-α 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.