Please use this identifier to cite or link to this item: http://hdl.handle.net/2440/74906
Type: Thesis
Title: The role of oxidant stress in a cellular model of aortic valve cell calcification.
Author: S. Kanagasingam, Navin
Issue Date: 2012
School/Discipline: School of Medicine
Abstract: INTRODUCTION: Calcific aortic valve stenosis (AS) is associated with a significant increase in morbidity and mortality in affected individuals, especially with advancing age. However, the pathogenesis of AS has not been fully understood, in particular, the role of oxidative stress and its contribution towards the development of AS. STUDY OBJECTIVE: The aim of the current study was to further delineate the role of redox stress, in particular as modulated by the endogenous anti-oxidant, thioredoxin (TRX) and the pro-oxidant, thioredoxin-interacting protein (TXNIP) following stimulation of cellular calcification / nodule formation induced by transforming growth factor-beta 1 (TGF-β1). In addition, the hypothesis that nitric oxide (NO) suppresses TXNIP expression in this system was also tested. METHODS: Cultured porcine aortic valve interstitial cells (AVICs) at 90% confluence were treated with TGF-β1 (5ng/ml) or vehicle, +/- 20μM Deta-NONOate (nitric oxide donor) or 10μM SB431542 (TGF-β1 inhibitor). TRX activity was quantified using the insulin disulphide reduction method with absorbance measured at 415 nm. Experiments were conducted in triplicate, and repeated in at least 3 cultures, between cell passages 2 and 4. Nodules were counted by an observer blinded to treatments. Experiments were also conducted in parallel, whereby TXNIP was measured by immunofluorescence and subsequently underwent image analysis. Cell survival quantification was performed in all experiments in response to various treatments as described above. Results were expressed as mean ± SEM. Multiple comparisons between the effects of treatments relative to respective controls were analyzed by one-way analysis of variance (ANOVA) with Bonferroni's multiple comparison test. A critical P<0.05 was considered statistically significant. RESULTS: TGF-β1 significantly increased calcific nodule formation compared to controls (37.19±2.67 vs. 0.33±0.12 (nodule count/well), P<0.001, n=4), and correspondingly decreased TRX activity (39.94±0.66 vs. 58.96±2.22 (mU/mg protein), P<0.001, n=4, figure 3.2) and cell survival/area (11.98±0.74 vs. 20.10±0.56 (x104/cm2), P<0.001, n=4), and increased TXNIP immunofluorescence (IF) intensity/cell (17059±204 vs. 7984±423 (arbitrary units), P<0.001, n=3). Deta-NONOate significantly suppressed TGF-β1-induced nodule formation (9.40±1.28 vs. 37.19±2.67 (nodule count/well), P<0.001, n=4), and correspondingly increased TRX activity (59.21±2.49 vs. 39.94±0.66 (mU/mg protein), P<0.001, n=4) and cell survival/area (16.93±0.95 vs. 11.98±0.74 (x10⁴/cm²), P<0.01, n=4), and decreased TXNIP IF intensity/cell (7918±310 vs. 17059±204 (arbitrary units), P<0.001, n=3), compared with TGF-β1 treatment alone. SB431542 significantly decreased TGF-β1-induced nodule formation (0.42±0.25 vs. 37.19±2.67 (nodule count/well), P<0.001, n=4), and correspondingly increased TRX activity (59.94±1.25 vs. 39.94±0.66 (mU/mg protein), P<0.001, n=3) and cell survival/area (20.50±0.78 vs. 11.98±0.74 ((x10⁴/cm²), P<0.001, n=4 ), and decreased TXNIP IF intensity/cell (7670±798 vs. 17059±204 (arbitrary units), P<0.001, n=3), compared with TGF-β1 treatment alone. CONCLUSION: TGF-β1-induced aortic valve interstitial cell calcific nodule formation is related to an increase in redox stress, involving a decrease in the endogenous antioxidant activity of thioredoxin (TRX), with a corresponding increase in the pro-oxidant, thioredoxin-interacting protein (TXNIP). In addition, TGF-β1-induced aortic valve interstitial cell calcific nodule formation results in a decrease in cell survival. These effects are ameliorated by nitric oxide (NO).
Advisor: Horowitz, John David
Kennedy, Jennifer Ann
Dissertation Note: Thesis (M.Med.Sc.) -- University of Adelaide, School of Medicine, 2012
Keywords: aortic stenosis; aortic sclerosis; valvular calcification; oxidative stress; nitric oxide
Appears in Collections:Research Theses

Files in This Item:
File Description SizeFormat 
01front.pdf50.63 kBAdobe PDFView/Open
02whole.pdf924.16 kBAdobe PDFView/Open


Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.