Title Page
Abstracts
Contents
Introduction 9
Materials and Methods 11
Reagent 11
Microarray data 11
Human subjects 11
VICs isolation and culture 11
Flowcytometric analysis 12
Cell viability and proliferation assay 12
Osteogenic differentiation 13
Autotaxin activity assay 13
Alkaline phosphatase (ALP) and alizarin red (AR) staining 13
Calcium assay 13
Quantitative real-time polymerase chain reaction (qPCR) 13
Enzyme-linked immunosorbent assay (ELISA) 14
Western blotting 14
Fluorescence reflectance imaging in vivo 14
Echocardiography 15
Immunohistochemistry 15
Mice 16
Rabbit 16
Statistical analysis 16
Results 20
ATX enzyme inhibitor reduces LPA production in VICs derived from FCAVD patients. 20
ATX inhibition attenuates the osteogenic differentiation of VICs in vitro. 21
ATX inhibitor downregulates both fibrosis and osteogenesis-related cytokine productions. 21
LPC-ATX-LPA axis promotes fibrosis via TGF-β signaling. 22
ATX inhibition suppresses calcific lesion formation in the mouse model. 23
FCAVD progression can be attenuated by ATX inhibitor in the rabbit model. 24
Discussion 26
References 42
국문요약 47
Table 1. Details of GEO datasets 18
Table 2. Primer sequences 19
Figure 1. Upregulation of ATX mRNA expression level in calcified human aortic valve tissue. 29
Figure 2. Identification of the isolated VICs by expression of surface markers. 30
Figure 3. Cytotoxicity of ATX inhibitor against VICs. 31
Figure 4. Effects of ATX inhibitor on FCAVD patient-derived VICs. 32
Figure 5. Effects of ATX inhibitor on osteogenic transdifferentiation and osteogenesis-related marker gene expressions in VICs. 33
Figure 6. Effects of ATX inhibitor on the production of cytokines related to inflammation, fibrosis, and osteogenesis in VICs. 34
Figure 7. Effects of ATX inhibitor on TGF-β signaling responses in VICs. 35
Figure 8. Effects of ATX inhibitor on TGF-β receptor expressions in VICs. 36
Figure 9. Effects of ATX inhibitor on CTGF expressions in VICs. 37
Figure 10. Effects of ATX inhibitor on fibrosis-related marker gene expressions in VICs. 38
Figure 11. Effects of ATX inhibitor on calcific lesion formation in a mouse FCAVD model. 39
Figure 12. Effects of ATX inhibitor on the aortic valve calcification in a rabbit FCAVD model. 40
Figure 13. Effects of ATX inhibitor on the fibrosis and calcification in the aortic valves of the rabbit FCAVD model. 41