Vascular calcification is present in most people over 60 years of age and results in severe complications. 1 Previous studies demonstrated that endothelial induction of Sox2 (SRY [sex-determining region Y]-box 2) triggered endothelial-mesenchymal transitions and drove endothelial cells toward osteoblastic differentiation. 1 The Sox2-positive endothelial cells invaded the aortic media to contribute to the calcific lesions. 1 Endothelial-specific deletion of Sox2 reduced endothelial-mesenchymal transitions and vascular calcification in matrix Gla protein null (Mgp−/−) mice, diabetic Ins2Akita/+ mice, and Apoe−/− mice fed a high fat diet. 1–3 Recently, we reported that β-adrenergic receptor antagonists (βblockers) decreased Sox2 expression in cerebrovascular endothelium and limited arteriovenous malformations. 4 Our results revealed that BMP (bone morphogenic protein) enhanced Notch signaling to induce Sox2. Depletion of the RBPJκ (recombination signal binding protein for immunoglobulin kappa J) abolished Sox2 induction. 5 We uncovered that pronethalol significantly reduced RBPJκ expression and its binding to Sox2 promoter, 5 thereby preventing excess BMP/Notch signaling from inducing Sox2. In this study, we hypothesize that the βblockers suppress Sox2 also in arterial endothelium to limit vascular calcification. To test this hypothesis, we treated Mgp−/− mice, a wellknown model of vascular calcification, 1 with pronethalol (20 mg/kg, intramuscular injection daily) for 2 weeks starting at 2 weeks of age. Wild-type mice and saline treatment were used as controls. We subsequently isolated CD31-expressing (CD31+) endothelial cells from the proximal descending aortas as previously reported1 and examined the expression of Sox2, Slug, and Osteopontin. Real-time polymerase chain reaction and immunoblotting showed that the pronethalol treatment abolished Sox2 induction (Figure [A]). Expression of Slug and Osteopontin, both regulated by Sox2 in Mgp−/− endothelium, 1 was also dramatically reduced (Figure [A]). Next, we examined the calcification in the proximal descending aortas from Mgp−/− mice. The total aortic calcium showed a robust decrease in the Mgp−/− aortas after pronethalol treatment (Figure [B]). Microcomputed tomography revealed that the pronethalol treatment significantly reduced the mineral density in Mgp−/− aortas (Figure [B]). Alizarin Red staining confirmed that pronethalol ameliorated the aortic calcification (Figure [B]). Immunostaining showed that Sox2 and Osteopontin was suppressed in the Mgp−/− aortic endothelium and media (Figure [B]). To examine the time-course of the pronethalol effect on calcification, we started the pronethalol treatment at 3 different time points, at 1, 2, and 3 weeks of age. The treatments all ended at 5 weeks of age. The results showed that the total aortic calcium was immediately reduced if the pronethalol was started at 1 or 2 weeks of age and was maintained at low levels as the treatment continued (Figure [C]). When the treatment was started at 3 weeks of age, it still reduced the aortic calcium but to a lesser degree (Figure [C]). We also examined the effect of pronethalol on bone formation in Mgp-/-mice. Microcomputed tomography showed no detectable differences between the mice (Figure [D]).