Endothelial phenotypes in a hypercholesterolemic swine model of early aortic valve sclerosis

Abstract

The aortic valve of humans and swine is preferentially susceptible to AVS localized to the aortic side of the leaflets. The endothelium may play a role in establishing this pattern of disease by regulating valve permeability, inflammation, and lipid transport in aortic valves, as it does in arteries. However, there are few experimental studies of valve endothelial cell and molecular biology. Consequently, valve endothelial homeostasis in general and the role of endothelium in early valve pathogenesis is poorly understood, particularly in relation to the preferential sidedness of AVS. Most challenging is the determination of spatially-defined valvular cell phenotypes in situ/in vivo. To study the transition from normal valve to AVS, I proposed that a brief in vivo exposure to hypercholesterolemia (HC), a well-established risk factor for AVS, will elicit differential side-specific changes in endothelial phenotypes, define their spatial heterogeneity and reveal molecular mechanisms underlying the initiation of aortic valve disease. A global genomics approach was chosen to address this hypothesis. I further proposed progressive changes in endothelial phenotypes during an extended 6 month HC period, studies that are addressed at the level of differential in situ protein expression. HC was induced in adult male swine over 2 weeks. Sub-endothelial lipid in-sudation and occasional calcific nodules were noted exclusively on the aortic side of the leaflets. Side-specific endothelial transcript profiles were determined using custom-printed porcine oligomer microarrays and investigated using bioinformatics and pathway analyses. Aortic-side and ventricular-side endothelia were highly heterogeneous in their responses to systemic HC; specifically, the aortic side endothelium displayed heightened sensitivity. HC induced the differential expression of 1325 endothelial genes on the aortic side in contrast to 87 genes on the pathoprotected ventricular side. Detailed pathway analyses identified multiple genes of the annexinA2-, caspase3-, PPARγ-, and TNFα-related pathways as differentially expressed on the aortic side in response to HC (vs normocholesterolemia) and, by independent analyses, relative to the ventricular side during HC. The directions of pathway gene differential expression were consistent with a protective endothelial phenotype. Protein immunocytochemistry substantiated the genomics results at 2 weeks and showed that differential HC-induced endothelial phenotype expression persisted at 6 months