Human degenerative valve disease is associated with up-regulation of low-density lipoprotein receptor-related protein 5 receptor-mediated bone formation

Abstract

Objectives: The goal of this research was to define the cellular mechanisms involved in myxomatous mitral valve disease and calcific aortic valve disease and to redefine the term degenerative valve disease in terms of an active cellular biology.

Background: "Degenerative" valvular heart disease is the primary cause of regurgitant and stenotic valvular lesion in the U.S. However, the signaling pathways are not known. We hypothesize that valve degeneration occurs due to an osteoblastic differentiation process mediated by the low-density lipoprotein receptor-related protein 5 (Lrp5) signaling pathway to cause valve thickening.

Methods: We examined human diseased valves: myxomatous mitral valves (n = 23), calcified tricuspid aortic valves (n = 27), calcified bicuspid aortic valves (n = 23), and control tissue from mitral and aortic valves (n = 40). The valves were examined by reverse transcriptase-polymerase chain reaction, Western blot, and immunohistochemistry for signaling markers important in osteoblast differentiation: Sox9 and Cbfa1 (transcription factors for osteoblast differentiation); Lrp5 and Wnt3 (osteoblast differentiation signaling marker), osteopontin and osteocalcin (osteoblast endochrondral bone matrix proteins), and proliferating cell nuclear antigen (a marker of cell proliferation). Cartilage development and bone formation was measured by Alcian blue stain and Alizarin red stain. Computed Scano MicroCT-40 (Bassersdorf, Switzerland) analysis measured calcium burden.

Results: Low-density lipoprotein receptor-related protein 5, osteocalcin, and other osteochrondrogenic differentiation markers were increased in the calcified aortic valves by protein and gene expression (p > 0.001). Sox9, Lrp5 receptor, and osteocalcin were increased in myxomatous mitral valves by protein and gene expression (p > 0.001). MicroCT was positive in the calcified aortic valves and negative in the myxomatous mitral valves.

Conclusions: The mechanism of valvular heart disease involves an endochondral bone process that is expressed as cartilage in the mitral valves and bone in the aortic valves. Up-regulation of the Lrp5 pathway may play a role in the mechanism for valvular heart disease.

Figure 1

Immunohistochemistry of the human mitral degenerative valves and calcified tricuspid and bicuspid aortic valves for calcification, cartilage, and MicroCT scan. Control valve, degenerative mitral valve (arrow points to hypertrophic chondrocytes), calcified aortic valves (arrow points to positive stain), and bicuspid aortic valve (arrow points to positive stain) (magnification 25×). (A) Alizarin red stain. (B) Alcian blue stain. (C) MicroCT scan (gray area is the soft tissue, white area is calcification, blue is the background).

Figure 2

Immunohistochemistry of the human mitral degenerative valves and calcified tricuspid and bicuspid aortic valves for non-collagenous bone matrix synthesis. Control valve, degenerative mitral valve (arrow points to hypertrophic chondrocytes), calcified aortic valves (arrow points to positive stain), and bicuspid aortic valve (arrow points to positive stain) (magnification 25×). Insert within each photo is a high-power magnification to demonstrate cellular staining (magnification 40×). (A) Bone sialoprotein stain. (B) Osteopontin stain. (C) Osteocalcin stain.

Figure 3

Immunohistochemistry of the human mitral degenerative valves and calcified tricuspid and bicuspid aortic valves for endochondral signaling markers low-density lipoprotein receptor-related protein 5/Wnt and proliferating cell nuclear antigen. Control valve, degenerative mitral valve (arrow points to hypertrophic chondrocytes), calcified aortic valves (arrow points to positive stain), and bicuspid aortic valve (arrow points to positive stain) (magnification 25×). Insert within each photo is a high-power magnification to demonstrate cellular staining (magnification 40×). (A) lipoprotein receptor-related protein 5 stain. (B) Wnt 3 stain. (C) Proliferating cell nuclear antigen stain.

Figure 4

Western blot. Western blot analysis for lipoprotein receptor-related protein 5 (Lrp5), osteocalcin, β-catenin, p42/44, and α-actin.

Figure 5

Semiquantitative reverse transcriptase-polymerase chain reaction. Reverse transcriptase-polymerase chain reaction results for Cbfa1, Sox9, cyclin, osteocalcin, and osteopontin.