Hypercholesterolemia induces side-specific phenotypic changes and peroxisome proliferator-activated receptor-gamma pathway activation in swine aortic valve endothelium

Abstract

Background- The endothelium of healthy aortic valves expresses different phenotypes on the aortic and ventricular sides. On the aortic side, which is susceptible to aortic valve sclerosis, there is a balanced coexpression of both propathological and protective pathways. Side-specific global gene expression can address endothelial phenotype balance in early aortic valve sclerosis.

Methods and results: Adult male swine were fed a hypercholesterolemic or an isocaloric normal diet for 2-week and 6-month periods. Hypercholesterolemia induced localized lipid insudation confined to the aortic side of the leaflet. Transcript profiling of valve endothelial populations showed that the susceptible aortic side was more sensitive to 2-week hypercholesterolemia than the ventricular side (1,325 vs 87 genes were differentially expressed). However, greater sensitivity was not evidence of a dysfunctional phenotype. Instead, pathway analyses identified differential expression of caspase 3-, peroxisome proliferator-activated receptor gamma-, TNF-alpha-, and nuclear factor-kappaB-related pathways that were consistent with a protective endothelial phenotype. This was confirmed at the protein level at 2 weeks and persisted at 6 months.

Conclusions: In a large animal model at high spatial resolution, endothelium on the pathosusceptible side of the aortic valve leaflet is responsive to hypercholesterolemia. Transcript profiles indicative of a protective phenotype were induced and persisted on the side prone to aortic valve sclerosis.

Figure 1

Aortic valve schematic illustrating aortic (black) and ventricular (white) side endothelium (a,upper panel). Hematoxylin and Oil Red O staining shows no lipid insudation in normal valves (a,lower panel); aortic side extra-cellular lipid insudation at 2-weeks HC (b); and increasing extra- and intra-cellular lipid deposition on the aortic side at 6-months HC (c).

Figure 2

Pathway analysis of genes up- (red) and down- (green) regulated in HC relative to NC aminals on the aortic side revealed clusters of highly inter-connected CASP3, TGF&[beta], ANXA2, and PPAR&[gamma]-centric genes. Particularly prominent were hypercholerolemia-responsive upstream regulators and downstream targets of PPAR&[gamma].

Figure 3

Pathway analysis of genes up- (pink) and down- (blue) regulated in A side endothelial cells relative to V side in aortic valves of HC swine. a. TNF&[alpha]-centric pathways were the highest scoring network of differentially expressed genes. b. TNF&[alpha] and NF&[kappa]B pathways were downregulated by hypercholesterolemia.

Figure 4

Side-specific expression of endothelial proteins in aortic valves exposed to HC for 2 weeks. a. PPAR&[gamma] expression was heterogeneous and confined to the A side endothelium. b. FABP1 was expressed in A side endothelial cells in a heterogeneous distribution.

Figure 5

Aortic side expression of PPAR&[gamma] pathway proteins in normal (NC), 2-week HC, and 6-month HC valves. There is minimal ABCA1, PPAR&[gamma], and FABP1 expression in normal valves. Upon exposure to HC, expression of these proteins is upregulated on the A side endothelium by 2-weeks and persisted at 6-months.