Antisense Oligonucleotide Targeting Sortilin Reduces Vascular Calcification in Mice in a Sex-Dependent Manner

BACKGROUND: Atherosclerotic calcification is a complex pathological process associated with an increased risk of cardiovascular events. Despite extensive research, this disorder has no effective therapeutic strategies. In this study, we evaluated the therapeutic potential of silencing Sort (sortilin) expression in vivo using antisense oligonucleotide (ASO-Sort1) to reduce the development of atherosclerosis and associated calcification. METHODS: Male and female Ldlr −/− (low-density lipoprotein receptor knock-out) mice were fed a high-fat diet and treated with ASO-Sort1 for 15 weeks. After ASO-Sort1 treatment, we evaluated atherosclerotic lesion formation and calcification through molecular imaging and histological techniques. The levels of plasma lipid and inflammatory molecules were determined. Unbiased liquid chromatography-mass spectrometry–based proteomics of aortic arches was conducted to investigate the sex-specific regulation in ASO-Sort1–treated mice. To validate our in vivo findings, we conducted in vitro experiments to examine whether β-estradiol treatment of mouse smooth muscle cells promotes calcification independently of Sort. RESULTS: ASO targeted primarily arterial smooth muscle cells, leading to a comparable reduction of aortic Sort1 expression by ≈57% in males and 52% in females. Although ASO-Sort1 did not affect the size of atherosclerotic lesions, it significantly reduced necrotic core development by 60% in male and 40% in female mice. In addition, it prevented aortic calcification by >50% only in male mice. Furthermore, proteome analysis revealed that while this treatment reduced vesicular trafficking, immune system, and extracellular matrix organization pathways in both male and female mice, it reduced autophagy-related processes specifically in males. In vitro results indicated that β-estradiol promotes calcification in smooth muscle cells treated with ASO-Sort1 by altering autophagy. CONCLUSIONS: Targeting Sort using antisense technology is effective in preventing vascular calcification in male mice. This unexpected outcome highlighted a novel sex-dependent discrepancy of the calcification pathway implicating the alteration of autophagy by β-estradiol and Sort.