Role for circulating osteogenic precursor cells in aortic valvular disease

Abstract

Objective: Approximately 13% of aortic valves removed from patients with end-stage aortic valve disease contain heterotopic ossification (HO). Recently, we identified a novel population of circulating osteogenic precursor (COP) cells that are derived from bone marrow and have the capability to form bone. These cells are identified by coexpression of the osteogenic marker type 1 collagen or osteoclacin and the hematopoietic marker CD45. We tested the hypothesis that these cells may contribute to heart valve stenosis.

Methods and results: Quantification of CD45(+) osteoclacin(+) COP cells by flow cytometry showed that they represent up to 1.1% of mononuclear cells. Clonally derived COP cells produce bone morphogenetic proteins 2 and 4 by immunohistochemical analysis. We reviewed 105 cases of end-stage aortic valvular disease and confirmed HO in 13 archived specimens. Using immunohistochemistry, we identified COP cells by coexpression of CD45 and type 1 collagen. There was a statistically significant association between the presence of COP cells and early HO lesions. COP cells were negligible in regions of unaffected valve leaflets (no HO) from the same individuals.

Conclusions: This study provides the first evidence that osteogenic cells in the blood home to sites of vascular injury and are associated with HO formation in heart valves.

Figure 1

COP cells are present in peripheral blood and are isolated as clonally-derived cell strains. (A) CD45⁺ OCN⁺ COP cells represent between 1.12% and 0.26% of circulating mononuclear cells. (B) Approximately 88 percent of clonally isolated and expanded CD45⁺ OCN⁺ COP cells are type 1 collagen positive.

Figure 2

Stages of endochondral ossification in aortic valve stenosis. Arrowheads indicate the border of fibroproliferative tissue and mature bone. Original magnification is 200X. Scale bar represents 5 microns.

Figure 3

COP cells are found in areas of fibroproliferation in affected ROIs. CD45⁺ Col1⁺ or CD45⁺ OCN⁺ COP cells are localized to regions of HO and dystrophic calcification, but not to unaffected regions. Original magnification is 200X. Scale bar represents 5 microns.

Figure 4

Perivascular localization of COP cells in affected ROIs. CD45⁺Col1⁺ or CD45⁺OCN⁺ COP cells are found near blood vessels (V) and are distinct from singly positive CD45 cells. Arrowheads indicate COP cells; arrows indicate singly positive CD45 cells. Original magnification is 200X. Scale bar represents 5 microns.

Figure 5

Putative role(s) of COP cells in the ossification of calcific aortic stenosis. ROS, reactive oxygen species; BMP, bone morphogenetic protein; SDF-1, stromal cell-derived factor-1; MFs, myofibroblasts; ?, possible derivation of myofibroblasts from COP cells.