Honors Program

University Honors

Date of Award

5-2013

Thesis Professor(s)

Douglas Thewke

Thesis Professor Department

Biochemistry and Molecular Biology

Thesis Reader(s)

Antonio Rusinol, Frank Hagelberg

Abstract

Introduction: Atherosclerosis is a chronic inflammatory disease characterized by the buildup of cholesterol, fat and other debris within arterial walls. Atherosclerotic lesions undergo a calcification process with similarities to bone remodeling. In mice, the type-2 cannabinoid receptor (CB2) is known to regulate bone remodeling processes and has also been shown to alter atherosclerotic lesion characteristics. However, the role of CB2 in lesion calcification is still unclear. CB2 modulates bone remodeling by affecting differentiation of osteogenic precursor cells; thus we hypothesize that CB2 alters lesion calcification by altering osteoblastogenesis and osteoclastogenesis of precursor cells of vascular origin. To test this hypothesis, we studied the role of CB2 receptor in mediating osteoclastogenesis and osteoblastogenesis from murine monocyte/macrophage and smooth muscle cell lines in vitro.

Methods: RAW264.7 cells are a murine monocyte/macrophage cell line known to undergo osteoclastogenesis in response to receptor activator of nuclear factor kappa B ligand (RANKL). RAW264.7 cells were cultured in media containing RANKL and supplemented with either CB2 agonists or antagonists. Effects on RANKL-induced osteoclastogenesis were then evaluated by measuring the osteoclast marker enzyme tartrate-resistant acid phosphatase (TRAP) activity and further verified by microscopic quantitation of multi-nucleate TRAP-positive osteoclasts. MOVAS-1 cells are a murine vascular aortic smooth muscle cell line known to differentiation into osteoblasts when cultured in osteogenic media. MOVAS-1 cells were cultured in osteogenic media supplemented with CB2 agonists or antagonists. Effects on osteoblastogenesis were evaluated by measuring marker enzyme activity. Alizarin red staining was performed to visualize and quantitate effects on calcium deposition.

Results: RAW264.7 cells treated with Win55, 212-2, a nonselective CB agonist, or HU-308, a selective CB2 agonist, displayed a dose-dependent decrease in RANKL-induced TRAP activity. Co-administration of a CB2-selective antagonist (SR144528), but not a CB1-selective antagonist (AM251), blocked this effect. Visual quantitation of multinucleated TRAP-positive cells confirmed Win55,212-2 treatment reduced osteoclastogenesis in RANKL-treated RAW264.7 cells. Induction of osteoblastic differentiation of MOVAS-1 cells, as determined by ALP activity, was enhanced by supplementation with Win55, 212-2 or 2-archidonyl glycerol. Co-administration of SR144528, but not AM251, reduced the induction of ALP activity in MOVAS-1 cells by Win55,212-2 and 2-AG. Alizarin red staining revealed increased calcium deposition in cultures of MOVAS-1 cells treated with Win55,212-2 compared to those cultured in osteogenic medium without Win55,212-2.

Conclusions: These results demonstrate that CB2 activation can affect osteogenic differentiation of vascular cells in vitro. These results support the hypothesis that CB2 signaling promotes lesion calcification by altering the balance of osteoclastic and osteoblastic differentiation of vascular precursors.

Document Type

Honors Thesis - Withheld

Copyright

Copyright by the authors.

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