Introduction: Aortic calcification is a significant pathological process associated with various cardiovascular diseases. While the sympathetic nervous system's influence on bone remodeling via β2-adrenergic receptors (β2-ARs) is well-established, its impact on vascular calcification remains relatively unexplored. Previous research suggests β2-ARs play a critical role in regulating valve calcification but have yet to be studied in aortic calcification. Thus, we aim to characterize the expression patterns of both β1-ARs and β2-ARs to elucidate the downstream signaling mediators in human aortic tissue and determine the effects of selective β2-AR agonists on aortic smooth muscle cell phenotype and function.

Methods: Calcified and non-calcified abdominal aortic wall segments from 6 patients with abdominal aortic aneurysm (AAA) were collected during elective open aortic repair at Northwestern Memorial Hospital (Chicago, IL). Samples were fixed, decalcified, and embedded in paraffin. Sections of 5 μm thickness were used for immunostaining of alpha-smooth muscle actin, β1-AR, and β2-AR. Human aortic smooth muscle cells (HAoSMC) were cultured in osteogenic media and treated with one of the following β-AR agonists (1 - 100 μmol/L): salmeterol, isoproterenol, norepinephrine, or an antagonist (0.3 – 30 μmol/L): ICI-118,551 or CGP-20712A. Cells were fixed for immunocytochemical staining of osteogenic markers (osteocalcin, osteopontin, and Runx2) and β-ARs. HAoSMCs were treated with salmeterol (1 μmol/L, β2-AR agonist) in the presence of either growth media or osteogenic media for up to 14 days. Metabolic and alkaline phosphatase (ALP) activity were measured with resazurin (n=8) and ALP assays (n=3), respectively, on days 7 and 14.

Results: Samples of abdominal aortic wall segments show differential expression of β2-adrenergic receptors between patients with abdominal aortic aneurysms. Culturing human aortic smooth muscle cells (hAoSMCs) in osteogenic (OST) media for 21 days successfully induced an osteogenic phenotype as evidenced by increased alkaline phosphatase (ALP) activity over time. Delivery of β2 agonist salmeterol to human aortic smooth muscle cells cultured in both growth media and osteogenic media showed no significant difference in metabolic activities after 21 days. Osteogenic cells treated with salmeterol demonstrate less alkaline phosphatase activity at day 21. Osteogenic differentiation of human aortic smooth muscle cells downregulates β2-adrenergic receptor expression. Stimulation of osteogenic smooth muscle cells with a β2 agonist, salmeterol, further downregulates receptor expression. Osteopontin expression decreased in osteogenic human aortic smooth muscle cells treated with salmeterol.

Conclusions: We observed a differential expression of β2-ARs across sections of human calcified aortas, suggesting a role of these receptors in areas subjected to different calcific stresses. In vitro experiments also demonstrated an impact on the calcification process. Further in vitro experiments with HAoSMCs treated with β-AR agonists and antagonists will help to elucidate the influence of β-adrenergic signaling on vascular calcification pathways.