Deletion of a Distal RANKL Gene Enhancer Delays Progression of Atherosclerotic Plaque Calcification in Hypercholesterolemic Mice

Receptor activator of NF-B ligand (RANKL) is a TNF-like cytokine which mediates diverse physiological functions including bone remodeling and immune regulation. RANKL has been identified in atherosclerotic lesions; however, its role in atherosclerotic plaque development remains elusive. An enhancer located 75kb upstream of the murine Rankl gene's transcription start site designated D5 is important for its calciotropic hormone- and cytokine-mediated expression. Here, we determined the impact of RANKL levels in atherosclerotic plaque development in the D5 enhancer-null (D5(-/-)) mice in an atherogenic Apoe(-/-) background fed a high-fat diet (HFD). Rankl mRNA transcripts were increased in aortic arches and thoracic aortae of Apoe(-/-) mice; however, this increase was blunted in Apoe(-/-);D5(-/-) mice. Similarly, higher Rankl transcripts were identified in splenic T lymphocytes in Apoe(-/-) mice, and their levels were reduced in Apoe(-/-);D5(-/-) mice. When analyzed by micro-computed tomography (mu CT), atherosclerotic plaque calcification was identified in six out of eight Apoe(-/-) mice, whereas only one out of eight Apoe(-/-);D5(-/-) mice developed plaque calcification after 12 weeks of HFD. However, following 18 weeks of HFD challenge, all of Apoe(-/-) and Apoe(-/-);D5(-/-) animals developed atherosclerotic plaque calcification. Likewise, atherosclerotic lesion sizes were site-specifically reduced in the aortic arch of Apoe(-/-);D5(-/-) mice at initial stage of atherosclerosis and this effect was diminished as atherosclerosis proceeded to a more advanced stage. Our data suggest that deletion of the RANKL D5 enhancer delays the progression of atherosclerotic plaque development and plaque calcification in hypercholesterolemic mice. This work provides important insight into RANKL's regulatory role in atherosclerosis. J. Cell. Biochem. 118: 4240-4253, 2017. (c) 2017 Wiley Periodicals, Inc.