Human aortic valve calcification is associated with an osteoblast phenotype

Abstract

Background: Calcific aortic stenosis is the third most common cardiovascular disease in the United States. We hypothesized that the mechanism for aortic valve calcification is similar to skeletal bone formation and that this process is mediated by an osteoblast-like phenotype.

Methods and results: To test this hypothesis, we examined calcified human aortic valves replaced at surgery (n=22) and normal human valves (n=20) removed at time of cardiac transplantation. Contact microradiography and micro-computerized tomography were used to assess the 2-dimensional and 3-dimensional extent of mineralization. Mineralization borders were identified with von Kossa and Goldner's stains. Electron microscopy and energy-dispersive spectroscopy were performed for identification of bone ultrastructure and CaPO4 composition. To analyze for the osteoblast and bone markers, reverse transcriptase-polymerase chain reaction was performed on calcified versus normal human valves for osteopontin, bone sialoprotein, osteocalcin, alkaline phosphatase, and the osteoblast-specific transcription factor Cbfa1. Microradiography and micro-computerized tomography confirmed the presence of calcification in the valve. Special stains for hydroxyapatite and CaPO4 were positive in calcification margins. Electron microscopy identified mineralization, whereas energy-dispersive spectroscopy confirmed the presence of elemental CaPO4. Reverse transcriptase-polymerase chain reaction revealed increased mRNA levels of osteopontin, bone sialoprotein, osteocalcin, and Cbfa1 in the calcified valves. There was no change in alkaline phosphatase mRNA level but an increase in the protein expression in the diseased valves.

Conclusions: These findings support the concept that aortic valve calcification is not a random degenerative process but an active regulated process associated with an osteoblast-like phenotype.

Figure 1

Histomorphometry of the calcified aortic valve. A1, X-ray microradiograph of the calcified valve. The smaller area of calcification is on the right, denoted by a star, and an arrow is pointing to the larger area of calcification on the left. A2, Star points to the Goldner’s-Masson Trichrome green stain for hydroxyapatite in the low magnification view. A3, Arrow points to the von Kossa black stain for calcium and phosphate in the high-magnification view. B1, MicroCT 2D reconstruction of the calcified aortic valve. B2, MicroCT 3D reconstruction of the calcified aortic valve.

Figure 2

Phenotype of the calcified aortic valve. A1, Electron dispersive spectroscopy confirms the presence of elemental CaPO₄. A2, Semiquantitative RT-PCR. RT-PCR using the total RNA for the bone markers demonstrating an increase in the RNA expression in all markers except alkaline phosphatase. B1, Immunogold electron microscopy. The arrow points to the alkaline phosphatase gold label in the calcified aortic valves, demonstrating a marked increase in label. B2, Electron microscopy. The arrow points to an area of mineralization in the valve (34K).