oa Calcification of Aortic Valve leaflets is Shear Dependent and Side-specific

Aortic valve (AV) sclerosis is a degenerative disease and is one of the leading causes of mortality in elderly population. AV experiences various mechanical stimuli such as pressure, shear and flexure with differing magnitudes on either sides of AV (fibrosa and ventricularis) with fibrosa side facing more dynamic environment. Normal hemodynamic conditions constantly renew and remodel the valve, whereas altered mechanical loading such as hypertension and altered shear stress can cause tissue inflammation that leads to calcification, preferentially on fibrosa side. AV calcification progressively leads to sclerosis and ultimately results in valve failure. However, the molecular and cellular processes that lead to inflammation and calcification are not very well understood. To understand the role of mechanics and underlying molecular mechanisms behind this preferential calcification, aortic side and ventricularis side of fresh porcine AVs were exposed to different shear stress patterns using an ex vivo cone and plate viscometer for 72 hours. Osteogenic medium was used to accelerate the calcification process ex vivo. To investigate the effect of shear stress magnitudes on fibrosa side, sine waveforms of amplitudes 5, 10 and 25 dynes/cm2 at a frequency of 1 Hz were used. To investigate the effects of shear stress frequency on fibrosa side, sine waveforms of 1 and 2 Hz at 10dynes/cm2 were used. Following exposure to shear, calcium levels of the samples were quantified using calcium Arsenazo assay. Von Kossa stain for mineralization was also done. Fresh porcine AVs were used as controls. Results indicated that low magnitude shear stress, 5 dyne/cm2 at 1 Hz elicited significant calcium levels on fibrosa side compared to other magnitudes and frequencies. To investigate if the oscillatory nature or the low magnitude was responsible for this high calcium response, fibrosa side was exposed to steady 5 dyne/cm2 under same experimental conditions as above. However, calcium levels at steady 5dyne/cm2 were comparable to fresh AV levels and thus non-significant. This result indicated that the magnitude in combination with the oscillatory nature of the sine 5dyne/cm2 triggered significant calcification levels on the fibrosa side of the AV leaflets. To test the side-specificity of this response, ventricularis side was also exposed to 5 dyne/cm2 under same experimental conditions as above. It is interesting to note that calcium levels on ventricularis side were not significant compared to that on the fibrosa side. This result further indicated that the expression of significant calcium levels in response to the low oscillatory shear is indeed side-specific. Thus our results suggest that the calcification of the AV leaflets is shear-dependent and side-specific. Shear dependent calcification in AV also suggests mechanobiological similarities with the atherosclerosis of blood vessels.