Development of Aortic Valve Disease in Familial Hypercholesterolemic Swine: Implications for Elucidating Disease Etiology

Abstract

Background: Familial hypercholesterolemia (FH) is a prevalent hereditary disease associated with increased atherosclerosis and calcific aortic valve disease (CAVD). However, in both FH and non-FH individuals, the role of hypercholesterolemia in the development of CAVD is poorly understood. This study used Rapacz FH (RFH) swine, an established model of human FH, to investigate the role of hypercholesterolemia alone in the initiation and progression of CAVD. The valves of RFH swine have not previously been examined.

Methods and results: Aortic valve leaflets were isolated from wild-type (0.25- and 1-year-old) and RFH (0.25-, 1-, 2-, and 3-year-old) swine. Adult RFH animals exhibited numerous hallmarks of early CAVD. Significant leaflet thickening was found in adult RFH swine, accompanied by extensive extracellular matrix remodeling, including proteoglycan enrichment, collagen disorganization, and elastin fragmentation. Increased lipid oxidation and infiltration of macrophages were also evident in adult RFH swine. Intracardiac echocardiography revealed mild aortic valve sclerosis in some of the adult RFH animals, but unimpaired valve function. Microarray analysis of valves from adult versus juvenile RFH animals revealed significant upregulation of inflammation-related genes, as well as several commonalities with atherosclerosis and overlap with human CAVD.

Conclusions: Adult RFH swine exhibited several hallmarks of early human CAVD, suggesting potential for these animals to help elucidate CAVD etiology in both FH and non-FH individuals. The development of advanced atherosclerotic lesions, but only early-stage CAVD, in RFH swine supports the hypothesis of an initial shared disease process, with additional stimulation necessary for further progression of CAVD.

Keywords: aortic valve; cardiovascular diseases; hypercholesterolemia.

Figure 1

Representative histology of atherosclerotic lesions in the coronary arteries of adult RFH swine. A, Hematoxylin and eosin (H&E) staining and Movat's Pentachrome revealed wall thickening, fibrin accumulation, and plaque buildup in coronary arteries. B, α‐SMA‐positive cells were detected in the subendothelial region. C, Lipid oxidation was observed by immunostaining of oxApoB and malondialdehyde. D, Apoptotic cells were identified throughout the arteries by immunostaining for caspase 3. E, Leukocyte degranulation (CD107a) and the secretion of MCP‐1 were also observed in the intima, as expected in an atherosclerotic plaque. Scale bars: 400 μm (A and C); 200 μm (B, D, and E). Arrows indicate areas representative of positive staining. N=4 animals. MCP‐1 indicates monocyte chemoattractant protein‐1; oxApoB, oxidatively modified apolipoprotein B; RFH, Rapacz familial hypercholesterolemic; α‐SMA, alpha‐smooth muscle actin.

Figure 2

Aortic valve leaflet microarchitecture in juvenile and adult RFH swine. Extensive remodeling of the extracellular matrix in 1‐ and 2‐year‐old RFH swine was observed by Movat's Pentachrome staining compared to juvenile RFH swine as well as both juvenile and 1‐year‐old WT swine. Specifically, adult RFH leaflets exhibited collagen disarray in layers other than the fibrosa, a substantial increase in proteoglycan content, and elastin fragmentation. Thickening of leaflets with age was also evident in RFH swine, whereas the juvenile and 1‐year‐old WT leaflets remained similar to each other in both architecture and thickness. N=3 to 4 animals/condition, n=3 samples/animal. Scale bar=400 μm. RFH indicates Rapacz familial hypercholesterolemic; WT, wild type.

Figure 3

Evaluation of adult RFH aortic valve function. ICE revealed that valve function was not impaired in any of the 3‐year‐old subjects (left panels). However, mild sclerosis was observed in two 3‐year‐old swine (right panels, arrows point at thickened leaflets). N=5 animals. ICE indicates intracardiac echocardiography; RFH, Rapacz familial hypercholesterolemic.

Figure 4

Lipid oxidation in RFH aortic valve leaflets. A, WT and RFH sections were stained for 2 markers of lipid oxidation: oxApoB and MDA. Arrows point to areas representative of positive staining. B, Deposition of oxApoB, the main structural component of low‐density lipoprotein, was observed in both WT and RFH leaflets. However, increased oxApoB accumulation of was present in 1‐ and 2‐year‐old RFH valves. C, Further evidence of oxidative stress was present in 1‐ and 2‐year‐old RFH, but not in WT 1‐year‐old or RFH juvenile leaflets, as demonstrated by immunostaining for MDA, a product of lipid peroxidation. *P<0.0500 compared to WT 1‐year‐old swine, ^# P<0.0500. N=3 to 4 animals/condition, n=3 samples/animal. Scale bars=100 μm. MDA indicates malondialdehyde; oxApoB, oxidatively modified apolipoprotein B; RFH, Rapacz familial hypercholesterolemic; WT, wild type.

Figure 5

Macrophages infiltrate adult RFH aortic valve leaflets. A, Accumulation of inflammatory marker MCP‐1 was observed in 2‐year‐old RFH swine, but not in 1‐year‐old WT or RFH swine. B, Presence of MCP‐1 in 2‐year‐old RFH swine was increased ≈3‐fold relative to other RFH and WT animals. C, Macrophages were detected only in thickened leaflet areas in 2‐year‐old RFH swine, as indicated by positive staining for CD68 in areas with thickness >400 μm. N=3 to 4 animals/condition, n=3 samples/animal. Scale bars=100 μm. MCP‐1 indicates monocyte chemoattractant protein 1; RFH, Rapacz familial hypercholesterolemic; WT, wild type. *P < 0.0500 compared to WT 1‐year‐old swine.