Date of Award

Summer 8-31-2019

Degree Type

Thesis

Degree Name

MS Biology

Department

Biology

Advisor

Jessica Cottrell, Ph.D.

Committee Member

Angela Klaus, Ph.D.

Committee Member

Constantine Bitsaktsis, Ph.D.

Keywords

osteoarthritis, chondrocyte, chondrogenesis, ossification, ABT-761, ATDC5

Abstract

5-Lipoxygenase metabolizes arachidonic acid (AA) to generate leukotrienes (LTs). Leukotrienes are very potent inflammatory mediators that can have negative effects on bone healing and cartilage tissue generation. Recent studies have shown that decreasing 5-lipoxygenase (5-LO) activity during fracture healing accelerates endochondral ossification and decreases healing time in animal fracture models, however, the roles of 5-LO in chondrogenesis is not fully investigated. In this study, we tested the hypothesis that 5-LO inhibition will increase the process of chondrogenesis. The ABT-761 is an N-hydroxyurea analog structurally similar to zileuton, which is a 5-LO enzyme inhibitor, The ATDC5 cell line which is derived from teratocarcinoma cells of mice is a chondrogenic cell line. The ATDC5 cells were treated with ABT-761 and Insulin. Then, they were evaluated for cellular proliferation via colorimetric assay, 5-LO gene expression via Immunocytochemistry (ICC) and Immunoblotting (Western Blot), and chondrogenesis by measuring calcium deposition, proteoglycan synthesis via Alizarin Red stain and Alcian Blue stain respectively at specific timepoints 7 days, 14 days, 21 days and 28 days. Our data demonstrated that ATDC5 cells express a 5-LO protein which can be directly inhibited by ABT-761 treatment. At a concentration of 10 μM, ABT-761 treatment does not affect ATDC5 cellular proliferation, however, higher doses inhibit significantly. In term of chondrogenesis, in this study, we demonstrated that 1μM ABT-761 has no effect on chondrogenesis, neither increase nor decrease, in ATDC5 chondrogenic cell line. In contrast, concentrations of 10 μM and 100μM ABT-761 have negative effects on chondrogenesis through which ABT-761 promotes chondrocyte apoptosis that may potentially lead to accelerating endochondral ossification or long bone formation in patients with a long bone fracture. However, ABT-761 may not be a clinical value to regenerate or promote chondrogenesis in patients suffering from osteoarthritis.

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