Abstract 3: Cholesterol Crystals Produced by Endothelial Cells During Hyperlipidemia Initiate Atherogenesis

Abstract

Atherosclerosis is caused by prolonged dyslipidemia and chronic inflammation. Although much is known about advanced stages of atherosclerosis, events leading to its initiation are poorly understood. Cholesterol crystals (CC) have been associated with advanced plaque, but their origin and potential role in the initiation of atherosclerosis and early plaque development are unknown. Using polarized light, scanning and transmission electron microscopy we show for the first time that CC are present in the very early plaque induced after only 1 week of high fat diet in ldlr-null mice. We determined that the source of CC is the endothelial monolayer by treatment of human aortic endothelial cells with LDL for 5 days. Other cell types like monocytes, fibroblasts and human umbilical vein endothelial cells failed to form CC upon LDL, AcLDL or oxLDL treatment. To identify in vitro formed CC we used polarized light, transmission electron and confocal microscopy. Furthermore, we identified that CC are generated within autophagolysosomes of the endothelium. The formation of CC by endothelial cells is accompanied with changes in intracellular cAMP levels. The consequences of early subendothelial CC formation are incomplete junction formation with subsequent impaired endothelial barrier integrity, processes known to be involved in atherosclerosis initiation and progression. Interestingly, increase of intracellular cAMP inhibited endothelial CC production in vitro and in vivo. By performing western Blot, flow cytometry and various microscopy techniques we could show that the inhibition of CC-formation as the results of cAMP increase is accompanied by a decrease autophagolysosomes as the CC producing organelles. We could furthermore demonstrate that this protective effect of cAMP could be inhibited by additional application of an eNOS inhibitor. In summary, we could demonstrate that CC are present in very early stages of atherosclerosis. These early subendothelial CC depositions are produced by autophagolysosomes within the endothelial monolayer in a cAMP/eNOS dependent manner. With these findings we might have found the so far missing link to initiation of plaque development in atherosclerosis.