Pronethalol Reduces Sox2 to Ameliorate Vascular Calcification

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 SRY (sex determining region Y)-box 2 (Sox2) triggered endothelial-mesenchymal transitions (EndMTs) and drove endothelial cells (ECs) towards osteoblastic differentiation ¹. The Sox2-positive ECs invaded the aortic media to contribute to the calcific lesions ¹. Endothelial-specific deletion of Sox2 reduced EndMTs and vascular calcification in matrix Gla protein null (Mgp^−/−) mice, diabetic Ins2^Akita/+ mice and Apoe^−/− mice fed a high fat diet ^1-3. Recently, we reported that beta-adrenergic receptor antagonists (beta-blockers) decreased Sox2 expression in cerebrovascular endothelium and limited arteriovenous malformations ⁴. Our results revealed that bone morphogenic protein (BMP) enhanced Notch signaling to induce Sox2. Depletion of the recombination signal binding protein for immunoglobulin kappa J (RBPJκ) 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 beta-blockers suppress Sox2 also in arterial endothelium to limit vascular calcification.

To test this hypothesis, we treated Mgp^−/− mice, a well-known model of vascular calcification ¹, with pronethalol (20 mg/kg, I.M. daily) for two weeks starting at 2 weeks of age. Wild type mice and saline treatment were used as controls. We subsequently isolated CD31-expressing (CD31+) ECs from the proximal descending aortas as previously reported ¹, and examined the expression of Sox2, Slug and Osteopontin. Real-time PCR and immunoblotting showed that the pronethalol treatment abolished Sox2 induction (Figure 1A). Expression of Slug and Osteopontin, both regulated by Sox2 in Mgp^−/− endothelium ¹, was also dramatically reduced (Figure 1A).

Figure 1: Pronethalol reduces Sox2 expression to ameliorate vascular calcification.

A, Expression of Sox2, Slug, Osteopontin (OPN) in CD31+ aortic ECs of Mgp^−/− mice with pronethalol treatment, as shown by real-time PCR and immunoblotting with densitometry (n=5). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a control gene for real-time PCR. Gender includes both biological and non-biological sex. The studies were approved by the Institutional Review Board and conducted in accordance with the animal care guidelines set by the University of California, Los Angeles.

B, Total calcium (top, left), micro-CT images (top, right), Alizarin Red staining (middle) and immunostaining (bottom) of Sox2 and osteopontin (OPN) (n=5). Scale bar, 2 mm (top); 100 μm (middle); 50 μm (bottom). Dashed circles indicate aortic tissues; red arrow points to the calcification.

C, Total calcium of the proximal descending aortas of Mgp^−/− mice. All the data were calculated by normalized to the calcium level on postnatal day 1. Asterisks indicate statistical significance of the comparisons between two consecutive treatments.

D, Micro-CT imaging and relative bone volume of Mgp^−/− mice treated with pronethalol (n=3). Scale bar, 0.5 mm

E, Oil Red O and Von Kossa staining of aortic sinus sections and total aortic calcium of the proximal descending aortas of fat-fed Apoe^−/− mice with pronethalol treatment (n=5). Scale bar, 100 μm; red arrows indicate calcification.

F, Alizarin Red staining and total calcium of the proximal descending aortas of Ins2^Akita/+ mice with pronethalol treatment (n=5).

Figure A, B and C were analyzed for statistical significance by ANOVA with post hoc Tukey’s analysis. Figure D, E and F were analyzed for statistical significance by unpaired Student's T-test. Error bars are mean ± standard deviation (SD). *, P<0.05; ***, P<0.001.

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 1B). Micro-computed tomography (Micro-CT) revealed that the pronethalol treatment significantly reduced the mineral density in Mgp^−/− aortas (Figure 1B). Alizarin Red staining confirmed that pronethalol ameliorated the aortic calcification (Figure 1B). Immunostaining showed that Sox2 and Osteopontin was suppressed in the Mgp^−/− aortic endothelium and media (Figure 1B). To examine the time-course of the pronethalol effect on calcification, we started the pronethalol treatment at three 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 1C). When the treatment was started at 3 weeks of age, it still reduced the aortic calcium but to a lesser degree (Figure 1C). We also examined the effect of pronethalol on bone formation in Mgp^−/− mice. Micro-CT showed no detectable differences between the mice (Figure 1D).

To determine if pronethalol limits vascular calcification also in atherosclerosis and diabetes, we first treated Apoe^−/− mice with pronethalol (20 mg/kg, I.M. daily). The treatment was started after 8 weeks on a high fat diet and lasted 4 weeks, during which the high fat diet was continued. Aortic calcium and von Kossa staining showed that pronethalol reduced the atherosclerotic lesion calcification (Figure 1E). We then treated Ins2^Akita/+ mice, a model of diabetic vascular calcification, with pronethalol (20 mg/kg, I.M. daily) for 4 weeks starting at 34 weeks of age. Aortic calcium and Alizarin Red staining showed that pronethalol decreased the calcification in Ins2^Akita/+ mice (Figure 1F). Together, the results suggest that the beta-blocker pronethalol reduces Sox2 expression in the arterial endothelium, thereby limiting EndMTs and vascular calcification.