Early Aberrant Angiogenesis Due to Elastic Fiber Fragmentation in Aortic Valve Disease

Abstract

Elastic fiber fragmentation (EFF) is a hallmark of aortic valve disease (AVD), and neovascularization has been identified as a late finding related to inflammation. We sought to characterize the relationship between early EFF and aberrant angiogenesis. To examine disease progression, regional anatomy and pathology of aortic valve tissue were assessed using histochemistry, immunohistochemistry, and electron microscopy from early-onset (<40 yo) and late-onset (≥40 yo) non-syndromic AVD specimens. To assess the effects of EFF on early AVD processes, valve tissue from Williams and Marfan syndrome patients was also analyzed. Bicuspid aortic valve was more common in early-onset AVD, and cardiovascular comorbidities were more common in late-onset AVD. Early-onset AVD specimens demonstrated angiogenesis without inflammation or atherosclerosis. A distinct pattern of elastic fiber components surrounded early-onset AVD neovessels, including increased emilin-1 and decreased fibulin-5. Different types of EFF were present in Williams syndrome (WS) and Marfan syndrome (MFS) aortic valves; WS but not MFS aortic valves demonstrated angiogenesis. Aberrant angiogenesis occurs in early-onset AVD in the absence of inflammation, implicating EFF. Elucidation of underlying mechanisms may inform the development of new pharmacologic treatments.

Keywords: angiogenesis; aortic root; elastic fiber; heart valves; pediatrics.

Figure 1

Aberrant angiogenesis in early-onset AVD precedes inflammation and atherosclerosis. Representative sections from control (left), early-onset AVD (center), and late-onset AVD (right) specimens. Normal elastic fiber structure is localized to the ventricularis layer of the cusp (arrow, A). Both early- and late-onset AVD are characterized by EFF and dispersion (B,C). Provisional (arrowhead) and overt (arrow) angiogenesis (D) is seen in early- (E) and late-onset (F) AVD, characterized in part by endothelial markers around neovessels (H,I) not observed in controls (G). Angiostatic CHM (J,K) is decreased in areas of angiogenesis in late-onset AVD (asterisk, L). Nominal CD-68 is seen in early-onset AVD (N), similar to control (M), corresponding with no atherosclerosis as demonstrated by absent LRP-5 staining (P,Q), both of which were markedly increased in late-onset AVD (double asterisks, O,R). The ventricularis cusp layer is oriented at the bottom of all panels.

Figure 2

Specific elastic fiber components are associated with overt angiogenesis. Representative sections from control (left column, A,C,E,G,I,K) and early-onset AVD (right column, B,D,F,H,J,L) specimens. In control tissue, elastin and fibulin-5 are localized in the cusp ventricularis layer (arrows), but fibrillin, emilin, fibulin-4, and LOX are also expressed weakly in all layers (arrowheads). In AVD tissue, elastin, fibrillin, emilin, and fibulin-4 are associated with neovessels (asterisk, B,D,F,H), whereas fibulin-5 and LOX are not (J,L).

Figure 3

Elastic fiber dysregulation impacts regional aortic valve structure. Regional aortic valve tissue (annulus, ring spongiosa, cusp) from control (A), early-onset AVD (B), WS (C), and MFS (D) specimens demonstrate specific differences in matrix organization. EFF in WS and MFS is different (arrows, C vs. D). Neovessels are seen in WS (arrowhead, C) but not MFS.

Figure 4

Ultrastructure of the valve annulus and cusp regions in WS and MFS. Collagen and elastic fiber organization in annulus (A–C) and cusp (D–F) regions from control (A,D), WS (B,E), and MFS (C,F) specimens. Cross sections of collagen fibril size and organization are demonstrated (insets). In control specimens, organized elastic fiber filaments are seen in the cusp (arrows) but not the annulus. WS specimens are characterized primarily by intra-EFF (B,E), whereas MFS has more inter-EFF (data not shown). There is proteoglycan accumulation in WS specimens (arrowheads, (B),(E)) and a “moth-eaten” edge appearance in MFS specimens (arrowheads, (C) and (F)). Collagen fibrils are small, irregular, and decreased in number in the cusp region of WS and the annulus region of MFS (asterisks, (C) and (E)).

Figure 5

Model of early EFF and angiogenesis preceding inflammation in AVD. Intact elastic fibers are organized as filaments in the normal ventricularis cusp layer (wavy black lines, A). Faulty elastic fiber assembly and/or imbalance between elastases and elastase inhibitors results in EFF (jagged black lines, (B). EFF causes angiogenesis (red circles), increased cell proliferation, and matrix accumulation (C), ultimately leading to inflammation (purple stars) and calcification (black nodules, D).