Spontaneous development of sclerotic changes of subchondral bone and cartilage degradation in endothelin-1 overexpression transgenic mice upon high fat/high cholesterol diet

Abstract

PURPOSE: Osteoarthritis has been found to be well correlated to metabolic syndromes like hypertension and obesity but being a multifaceted degenerative disease, many of the actual biochemical pathways remain unsolved. We have previously demonstrated the effect of endothelin-1, a potent vasoconstrictor that contributes to hypertension, in the development of osteoarthritic changes in the subchondral bone. This time, we would like to investigate the interplay and biochemical roles of obesity and endothelin-1 in osteoarthritis and propose the use of obese endothelin-1 overexpressing mice as an OA model. METHODS: Male, 35-week-old endothelin-1 overexpressing (TET) mice and their non-transgenic littermates were used in this study. Half of each group was fed with high fat, high cholesterol chow to induce obesity, and the other half was fed with normal chow. Knee joints were harvested and fixed in 10% formalin solution immediately after sacrifice. Micro-CT scans of the knee joints were performed before tissue processing. Subchondral bone structures were analysed in CTAn Software. Samples were embedded for paraffin sectioning at 5μm at sagittal plane. Safranin-O staining was done to evaluate the cartilage degeneration indicated by loss of glycosaminoglycan content. Immunohistochemical staining was performed using rabbit primary antibodies coupled with Streptavidin-HRP developed with DAB substrate, and counterstained with Harris Haematoxylin. RESULTS: TET mice fed with high fat & high cholesterol diet were found to have higher bone volume fraction (43.3% vs 36.3%) and lower bone surface to volume ratio (34.66% vs 44.5%) when compared to those fed with normal chow. We have also found a significant difference (p=0.01) in trabecular thickness across the four test groups. From the IHC staining, there was enhanced osterix expression, indicating more bone formation in the TET groups. From the Tatrate Resistant Acid Phosphatase (TRAP) staining, more active osteoclasts were observed. There was also cartilage proteoglycan loss as indicated by the Safranin-O staining. CONCLUSIONS: We have observed remarkable sclerotic changes in secondary spongiosa in TET mice fed with high fat, high cholesterol diet when compared to the other three groups, including increased bone volume fraction, trabecular thickness, intersection surface and decreased bone surface to volume ratio. Similarly, our staining results also suggest higher bone resorption and formation rate, i.e. higher remodelling rate. Neither the high fat high cholesterol diet nor the overexpression of endothelin-1 alone causes spontaneous osteoarthritic changes, but their combined effect leads to remarkable spontaneous changes in the subchondral region.