Dissolving cholesterol crystals reduces aortic valve stenosis development in mice
Authors: Niepmann S.T , Lenart M , Willemsen N , Duewell P , Luetjohann D , Latz E , Nickenig G , Zimmer S 
Published: November 2020

Abstract

Background
Aortic valve stenosis (AS) is the most common valve disease worldwide and is associated with a very high morbidity and mortality. Until today, aortic valve replacement is the only therapeutic option available. Analysis of explanted human aortic valves has shown that atherosclerosis-like lesions, that contain cholesterol crystals (CC), are present in stenotic aortic valve cusps. It has been demonstrated that CCs can activate the NLRP3 inflammasome and hereby trigger a complex, IL-1b driven, inflammatory response. 2-hydroxypropyl-β-cyclodextrin (CD) is a cyclic oligosaccharide that can increase the solubility of CCs, which results in a reduction of CC-load and therefore could inhibit the pro-inflammatory immune response.

Methods
Severe AS was induced in 10 weeks old C57BL/6-J (WT) and Apolipoprotein-E-deficient (ApoE) mice. Acoronary springwire was used to induce an endothelial injury under echocardiographic guidance. AS development was confirmed via ultrasound examinations. ApoE mice were fed a cholesterol-rich western diet and concomitantly received daily injections of 2g/kg/d CD via subcutaneous injection. CCs were visualized with laser reflection confocal microscopy. Serum cholesterol analysis were performed via mass GC-MS-SIM.

Results
In order to evaluate whether hyperlipidemia aggravates AS development, WT and ApoE mice were fed a cholesterol-rich diet and subjected to our model of wire-induced AS. Trans-aortic valve peak velocity levels of ApoE mice were significantly increased six weeks after injury compared to WT mice. Histological analysis of these mice showed large CC-deposits in the aortic valves of ApoE mice. Next, CC solubility was increased in a group of ApoE mice, control mice only received PBS injections. Interestingly, mice treated with CD displayed a significantly reduced peak blood velocity over the aortic valve compared to PBS mice. Left ventricular ejection fraction remained unchanged. Serum cholesterol analysis was performed to analyze the effect of CD on cholesterol metabolism. 27-hydroxycholesterol, an endogenous oxysterol of cholesterol metabolism, which reduces the potential for the conversion of free cholesterol into crystals was significantly increased in CD treated mice. The levels of cholesterol precursors were unchanged, indicating that CD doesn't influence de-novo synthesis of cholesterol. Intestinal absorption of cholesterol was also not affected by CD, as assessed by quantification of phytosterols in the serum of CD and PBS treated mice.

Conclusion
These results underline the importance of hyperlipidemia in the pathogenesis of AS. Particularly CCs seem to act as an important inflammatory trigger in the development of AS. Increasing the solubility of cholesterol through CD reduces AS development in mice and could, as it is already considered safe in human, act as a possible therapeutic option.

Funding Acknowledgement
Type of funding source: Public grant(s) – National budget only. Main funding source(s): DFG, German Research Foundation