Circulating Osteogenic Cells in End-Stage Aortic Valvular Disease

1. Abstract Approximately 13 percent of heart valves removed from patients with end-stage aortic valve disease contains heterotopic ossification (HO). Recently we identified a novel population of circulating osteogenic precursor (COP) cells which are derived from bone marrow and have the capability to form bone (Suda et al., Stem Cells 27:2209-2219, 2009).These cells are identified by the coexpression of the osteogenic marker type 1 collagen and the hematopoietic marker CD45. High levels of COP cells are strongly associated with HO formation in genetic and non-genetic conditions and they can be localized to early fibroproliferative lesions. We tested the hypothesis that these cells may contribute to heart valve stenosis and examined their presence in surgically removed heart valves containing HO. 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 type 1 collagen and CD45. There was a statistically significant association between the presence of COP cells and early lesions of HO. COP cells were negligible or absent in regions of unaffected valve leaflets (no HO) from the same individuals. This study provides evidence that osteogenic cells in the blood home to sites of vascular injury and are associated with HO formation in heart valves with advanced disease. Figure 3. Type 1 collagen staining is colocalized with CD45 staining in COP cells. Immunohistochemistry staining for collagen (green) and CD45 (red), analyzed by confocal laser-scanning microscopy. Original magnification 630X Background The pathogenesis of aortic valve stenosis involves active processes of inflammation, changes in lipid metabolism, dystrophic calcification and unexpected bone formation1. The aortic valve undergoes continuous repair in order to remain pliable, and aberrant tissue remodeling with age results in the accumulation of calcium (hydroxyapatite mineral), sclerotic tissue, and ultimately heterotopic ossification (HO) through an endochondral mechanism2. Mature lamellar bone formation, bone remodeling, and even microfracture repair occurs in the end-stage stenotic valve2. The cells responsible for ectopic bone formation in end-stage disease are unknown3. Recently, we described circulating osteogenic precursor (COP) cells as a bone marrow-derived type 1 collagen+/CD45+ subpopulation of mononuclear adherent cells that are present in early pre-osseous lesions of HO4. These cells were able to nucleate extra-skeletal ossification in a murine in vivo implantation assay4. That circulating, hematopoietic-derived cells with osteogenic potential can seed inflammatory sites suggested the possibility of their involvement in end-stage aortic valve disease. Approximately 13 percent of heart valves removed from patients with end-stage aortic valve disease contain HO. Here, we tested the hypothesis that COP cells may contribute to heart valve stenosis and examined their presence in surgically removed aortic heart valves containing HO. Figure 1. COP cells are found within areas of fibroproliferation and neovascularity. (A) A stenotic heart valve with mature bone elements, including osteocytes (white arrow heads) and bone lining cells (black arrow heads). In regions of fibroproliferation and neovascularity (B), COP cells (orange) were identified by co-expression of type 1 collagen (green) and CD45 (red) staining (C). Original magnification 200X. A and B, Hemotoxylin & eosin staining; C, Immunofluorescence of specific markers as described in Materials & Methods. Materials and Methods Patients. Archived tissue blocks were obtained in accordance with institutional review board approval at the University of Pennsylvania. Heart valves were excised (owing to valvular stenosis or regurgitation) during cardiac valve replacement surgery between 1994 and 1998 at the University of Pennsylvania Health System2. One hundred and five cases were reviewed for pathological evaluation of cardiac valves and patient demographic information. Histological evaluation of heart valves. Formalin-fixed tissue specimens undergoing limited demineralization after 10% formic acid treatment were sectioned at a depth of five microns for histological analysis. Standard H & E staining of sections was performed to identify mature lamellar bone and only heart valves confirmed to contain bone were included in the study. Regions of interest (ROIs) within each heart valve were defined by arbitrary division of each leaflet into quarter sections for evaluation of the presence of ectopic ossification. Immunohistochemistry. Heat-induced epitope retrieval at pH 6.0 in 10 mM sodium citrate buffer was performed on all sections. After heating to 95° C for 20 min, sections were then allowed to cool to room temperature in the buffer. Sections were blocked using 20% serum. Primary antibodies against CD45 (Santa Cruz) and type-1 collagen (Fitzgerald Industries) were diluted to 1:100 and incubated with sections for 18 h in a dark humidity chamber at 4°C. Following primary antibody incubation, sections were washed twice for 5 min in deionized water with 0.2% Tween-20. Secondary antibodies against mouse and rabbit IgGs, tagged with Alexa 555 and Alexa 647 fluorescent labels, respectively, were incubated at a concentration of 1:1000 in a dark humidity chamber for 60 min. After washing twice for 5 min with 0.2% Tween-20, 4',6-diamidino-2-phenylindole (DAPI) was incubated with sections for 5 min, followed by a final wash. Summary • By immunohistochemistry, we identified COP cells in end-stage aortic heart valves by coexpression of type 1 collagen and CD45. • COP cells were found in early lesions of heterotopic ossification (HO) in all of the samples examined, but were negligible or absent in regions of unaffected valve leaflets (no HO) from the same individuals. • This study provides evidence that osteogenic cells in the blood home to sites of vascular injury and are associated with HO formation in stenotic heart valves. References • Mohler ER 3rd. Mechanisms of aortic valve calcification. Am J Cardiol 2004; 94:1396-402. • Mohler ER 3rd, Gannon F, Reynolds C, Zimmerman R, Keane MG, Kaplan FS. Bone • formation and inflammation in cardiac valves. Circulation 2001; 103:1522-8. • 3. Rajamannan NM, Subramaniam M, Rickard D, Stock SR, Donovan J, Springett M, et al. • Human aortic valve calcification is associated with an osteoblast phenotype. Circulation • 2003; 107:2181-4. • 4. Suda RK, Billings PC, Egan KP, Kim JH, McCarrick-Walmsley R, Glaser DL, et al. • Circulating osteogenic precursor cells in heterotopic bone formation. Stem Cells 2009; • 27:2209-19. Figure 2. Col1+ CD45+ COP cells are found in affected regions (containing HO) of stenotic aortic valve leaflets. Regions unaffected (top panel) and affected (bottom panel) by HO are shown. Immunohistochemistry staining for type 1 collagen (green) and CD45 (red) is as shown. Original magnification 200X. Microscopy. Sections were viewed with a Nikon Eclipse TE-2000U inverted fluorescent microscope attached to a Photometrics Cool snap camera using NIS-elements Ar version 3.0. Emission times were standardized to the negative control slides. Confocal analysis was performed on a Zeiss LSM510 META NLO laser scanning confocal microscope running Zeiss LSM510META version 3.2 at the University of Pennsylvania Biomedical Imaging Core. Quantitation of COP cells in tissue specimens. At least 200 cells (DAPI positive) were scored in areas of fibroproliferative tissue for Col 1+ CD45+ cells. Cells were scored in ROIs containing affected (HO present) or unaffected areas. Unaffected ROIs from the aortic valve of the same individual served as negative controls. Statistical Analysis. The t test (Student’s t test; two-sided and paired) was used to determine whether the mean value for the number of COP cells (continuous variable) in the affected ROIs differed significantly from those in the unaffected (control) ROIs. An unpaired Student’s t test (two-sided) was used to determine if the mean number of COP cells in affected ROIs in male-derived valves was significantly different from affected ROIs in female-derived valves. Statistical significance was set to p = 0.05. All statistics were performed by Graphpad software (www.graphpad.com). Circulating Osteogenic Cells in End-Stage Aortic Valvular Disease Results Kevin P. Egan, BA1and Robert J. Pignolo, MD, PhD11Division of Geriatrics, Department of Medicine, University of Pennsylvania, Philadelphia, PA