Role of circulating osteogenic progenitor cells in calcific aortic stenosis

Abstract

Objectives: The purpose of this study was to determine the role of circulating endothelial progenitor cells with osteoblastic phenotype (EPC-OCN) in human aortic valve calcification (AVC).

Background: Recent evidence suggests that rather than passive mineralization, AVC is an active atherosclerotic process with an osteoblastic component resembling coronary calcification. We have recently identified circulating EPCs with osteogenic properties carrying both endothelial progenitor (CD34, KDR) and osteoblastic (osteocalcin [OCN]) cell surface markers.

Methods: Blood samples from controls (n = 22) and patients with mild to moderate calcific aortic stenosis (mi-moAS, n = 17), severe calcific AS (sAS, n = 26), and both sAS and severe coronary artery disease (sCAD) (n = 33) were collected during diagnostic coronary angiography. By using flow cytometry, peripheral blood mononuclear cells were analyzed for CD34, KDR, and OCN. Resected normal and calcified aortic valves were analyzed histologically.

Results: Patients with mi-moAS and patients with sAS/sCAD had significantly less EPCs (CD34+/KDR+/OCN-) than controls. Patients with sAS showed significantly higher numbers of EPC-OCN (CD34+/KDR+/OCN+) than controls. In addition, the percentage of EPC costaining for OCN was higher in all disease groups compared with controls. A subgroup analysis of younger patients with bicuspid sAS showed a similar pattern of significantly lower EPCs but a high percentage of coexpression of OCN. Immunofluorescence showed colocalization of nuclear factor kappa-B and OCN in diseased and normal valves. CD34+/OCN+ cells were abundant in the endothelial and deeper cell layers of calcific aortic valve tissue but not in normal aortic valve tissue.

Conclusions: Circulating EPC-OCN may play a significant role in the pathogenesis and as markers of prognostication of calcific AS.

Figure 1. Circulating EPC-OCN in All 4 Study Groups

Patients with mi-moAS and sAS showed higher numbers of circulating endothelial progenitor cells with osteoblastic phenotype (EPC-OCN) than normal subjects, whereas patients with both sAS and sCAD had numbers of circulating EPC-OCN similar to those of normal subjects. *p = 0.024 versus normal subjects. Median values and interquartile ranges are shown; diamonds represent mean values. CAD = coronary artery disease; mi-moAS = mild to moderate calcific aortic stenosis; sAS = severe calcific aortic stenosis; sCAD = severe coronary artery disease.

Figure 2. Percentage of OCN Costaining of EPCs in All 4 Study Groups

All disease groups showed a higher percentage of costaining of circulating EPCs for osteocalcin (OCN) than normal subjects, reaching statistical significance in all but the sAS group. *p < 0.001, #p = 0.043 versus normal subjects. Median values and interquartile ranges are shown; diamonds represent mean values. Abbreviations as in Figure 1.

Figure 3. Circulating EPCs in All 4 Study Groups

Patients with mi-moAS and patients with both sAS and sCAD showed significantly less circulating EPCs than normal subjects. *p < 0.001, #p = 0.003 versus normal subjects. Median values and interquartile ranges are shown; diamonds represent mean values. Abbreviations as in Figure 1.

Figure 4. Colocalization of Inflammation and Osteoblastic Activity

Immunofluorescence staining for nuclear factor kappa-B (NFkB) (red) and osteocalcin (OCN) (green) of control (A, B) and severely calcified aortic valves (C, D; blue represents DAPI stain). NFkB and OCN demonstrate colocalization (yellow, examples indicated by white arrowheads), indicating that the osteoblastic process is associated with inflammation. Whereas calcified aortic valves show NFkB/OCN costaining within the endothelial cell layer (C) and the deeper valve tissue layers (D), control valves show costaining only within the valve (B) and not within the endothelial cell layer (A). Magnification ×40. For individual color channels, please see Online Figures 1 and 2. AS = aortic stenosis.

Figure 5. Immunofluorescence Staining for Osteoblastic Progenitor Cells

Immunofluorescence staining for CD34 (red) and osteocalcin (OCN) (green) of normal (A, B) and severely calcified aortic valves (C, D; blue represents DAPI stain). CD34/OCN costaining (yellow, examples indicated by white arrow-heads) is rarely observed within deeper valve tissue of control valves (B), but not within the endothelial cell layer (A). In contrast, CD34/OCN cells are abundant within the endothelial cell layer (C) and deeper valve tissue (D) of severely calcified valves. Magnification ×40. For individual color channels, please see Online Figures 1 and 2. AS = aortic stenosis.

Figure 6. Possible Role of Circulating EPC-OCN in AVC

In the hypothesized interplay among the circulating EPC-OCN, the endothelial cell layers of the aortic valve, and the VICs, EPC-OCN may engraft into the endothelial cell layer or enter deeper valvular tissue layers and become VICs (see “Discussion” for further details). AVC = aortic valve calcification; EC = endothelial cell; EPC-OCN = circulating endothelial progenitor cells with osteoblastic phenotype; VIC = valve interstitial cell.