Panel A shows the forward scatter (FSC), side scatter (SSC), and the gates used in the analysis. Panel B shows the staining with the isotype controls for the CD34- and KDR-specific antibodies, and panel C shows the specific staining with these antibodies. Panel D shows the CD34⁺/KDR⁺ cells stained with the osteocalcin (OCN) antibody. We required that <0.5% of the cells were positive with the isotype controls, and for all data reported in the article, we subtracted the signal of the isotype from the signal due to the specific antibodies. APC = allophycocyanin; IgG = immunoglobulin G; KDR = kinase insert domain receptor.

(A) CD34⁺/KDR⁺/OCN⁺ cells, expressed as absolute counts per 100,000 counts, and (B) percentage of OCN⁺ of CD34⁺/KDR⁺ cells in control subjects (circles), patients with early coronary atherosclerosis (ECA, triangles), and in patients with late coronary atherosclerosis (LCA, squares). *p < 0.05 and **p < 0.01 compared with control subjects. Shown are the median values and interquartile ranges. Abbreviations as in Figure 1.

(A) CD34⁺/CD133⁺/KDR⁺/OCN⁺ cells, expressed as absolute counts per 100,000 counts, and (B) percentage of OCN⁺ of CD34⁺/CD133⁺/KDR⁺ cells in control subjects (circles), patients with ECA (triangles), and in patients with LCA (squares). *p < 0.05 and p = 0.071 compared with control subjects. Shown are the median values and interquartile ranges. Abbreviations as in Figures 1 and 2.

(A) CD34⁺/CD133^–/KDR⁺/OCN⁺ cells, expressed as absolute counts per 100,000 counts, and (B) percentage of OCN⁺ of CD34⁺/CD133^–/KDR⁺ cells in control subjects (circles), patients with ECA (triangles), and in patients with LCA (squares). *p < 0.05, **p < 0.01, and ***p < 0.001 compared with control subjects. Shown are the median values and interquartile ranges. Abbreviations as in Figures 1 and 2.

Correlation between serum matrix Gla protein (MGP) levels in the LCA patients and (A) CD34⁺/CD133^–/KDR⁺/OCN⁺ cells, expressed as absolute counts per 100,000 counts, and (B) percentage of OCN⁺ of CD34⁺/CD133^–/KDR⁺ cells. Abbreviations as in Figures 1 and 2.

Confocal microscopy showing a cell staining with all 4 antibodies used in the study (OCN, CD34, CD133, and KDR). Shown also is a stain for nuclei (4,6-diamino-2-phenylindole or DAPI) and the merged images. Abbreviations as in Figure 1.

In vitro mineralization assay of CD34⁺ cells from an anonymous blood donor using (A) von Kossa staining and (B) alizarin red staining, and (C) of peripheral blood mononuclear cells from a patient with LCA (alizarin red stain). In these cultures, cells were exposed to Ca²⁺ for 5 days followed by 21 days of culture in osteoblast differentiation medium. All images are at 4x magnification. Abbreviation as in Figure 2.

Gene expression analysis of CD34⁺ cells from 6 anonymous blood donors. (A) Expression of bone-related and other genes normalized to undifferentiated human bone marrow stromal cells (hMSCs; expression of each gene in hMSCs set at 1.0); (B) changes in expression of messenger ribonucleic acids (mRNAs) for CD34 and CD45 following in vitro culture; (C) changes in expression of mRNAs for osteopontin and Col1 alpha₁ following in vitro culture; and (D) changes in mRNA expression for Col1 alpha₁ following in vitro culture in the 3 samples with the highest versus the lowest expression of OCN mRNA prior to culture. *p < 0.05, **p < 0.01, and ***p < 0.001 compared with 1.0 using a 1-sample t test; p < 0.01 and p < 0.001 compared with freshly sorted CD34 cells using a 2-sample t test. N.D. = not detected; other abbreviation as in Figure 1.