Parathyroid hormone stimulates circulating osteogenic cells in hypoparathyroidism

Abstract

Context: The osteoanabolic properties of PTH may be due to increases in the number and maturity of circulating osteogenic cells. Hypoparathyroidism is a useful clinical model because this hypothesis can be tested by administering PTH.

Objective: The objective of the study was to characterize circulating osteogenic cells in hypoparathyroid subjects during 12 months of PTH (1-84) administration.

Design: Osteogenic cells were characterized using flow cytometry and antibodies against osteocalcin, an osteoblast-specific protein product, and stem cell markers CD34 and CD146. Changes in bone formation from biochemical markers and quadruple-labeled transiliac crest bone biopsies (0 and 3 month time points) were correlated with measurements of circulating osteogenic cells.

Setting: The study was conducted at a clinical research center.

Patients: Nineteen control and 19 hypoparathyroid patients were included in the study.

Intervention: Intervention included the administration of PTH (1-84).

Results: Osteocalcin-positive cells were lower in hypoparathyroid subjects than controls (0.7 ± 0.1 vs. 2.0 ± 0.1%; P < 0.0001), with greater coexpression of the early cell markers CD34 and CD146 among the osteocalcin-positive cells in the hypoparathyroid subjects (11.0 ± 1.0 vs. 5.6 ± 0.7%; P < 0.001). With PTH (1-84) administration, the number of osteogenic cells increased 3-fold (P < 0.0001), whereas the coexpression of the early cell markers CD34 and CD146 decreased. Increases in osteogenic cells correlated with circulating and histomorphometric indices of osteoblast function: N-terminal propeptide of type I procollagen (R(2) = 0.4, P ≤ 0.001), bone-specific alkaline phosphatase (R(2) = 0.3, P < 0.001), osteocalcin (R(2) = 0.4, P < 0.001), mineralized perimeter (R(2) = 0.5, P < 0.001), mineral apposition rate (R(2) = 0.4, P = 0.003), and bone formation rate (R(2) = 0.5, P < 0.001).

Conclusions: It is likely that PTH stimulates bone formation by stimulating osteoblast development and maturation. Correlations between circulating osteogenic cells and histomorphometric indices of bone formation establish that osteoblast activity is being identified by this methodology.

Figure 1

Representative example of flow cytometry dot plots looking at OCN⁺ population. The isotype control shows the nonspecific binding, or background noise. To measure the specific expression of OCN, the isotype control is subtracted from the total OCN. In the control, the OCN⁺ cells are 2.9–0.6% of circulating PBMCs (2.3%), whereas in the matched hypoparathyroid (Hypo) subject, the OCN⁺ cells are 1.2–0.3% of circulating PBMCs (0.9%).

Figure 2

Comparison of cell populations in untreated hypoparathyroid subjects and matched controls. Data are mean ± sem. A, Comparison of OCN⁺ cells. The percentage of PBMCs that was positive for OCN was lower in the 19 hypoparathyroid subjects compared with the 19 matched controls. B, Comparison of OCN⁺/CD34⁺ cells. The percentage of OCN⁺ cells that was also positive for CD34 showed a trend to be higher in the hypoparathyroid subjects. C, Comparison of OCN⁺/CD146⁺ cells. The percentage of OCN⁺ cells that was also positive for CD146 was higher in the hypoparathyroid subjects. D, Comparison of OCN⁺/CD34⁺/CD146⁺ cells. The percentage of OCN⁺ cells that was also positive for both CD34 and CD146 was higher in the hypoparathyroid subjects.

Figure 3

Changes in cell populations in hypoparathyroid subjects with PTH (1-84) treatment over 12 months. Data are mean ± sem. *, P < 0.05 from baseline; **, P < 0.001 from baseline. A, Change in OCN⁺ cells. The percentage of PBMCs that was OCN⁺ cells increased with PTH (1-84) treatment. B, Change in OCN⁺/CD34⁻ cells. The percentage of OCN⁺ cells that was lacking CD34 increased with PTH (1-84) treatment. C, Change in OCN⁺/CD34⁻/CD146⁻ cells. The percentage of OCN⁺ cells that was lacking both CD34 and CD146 increased with PTH (1-84) treatment. D, Time course of increase in the OCN⁺ cell population compared with time course of increase in biochemical markers of bone formation with PTH (1-84) treatment.

Figure 4

A representative quadruple-label biopsy from a 25-yr-old hypoparathyroid woman before and after PTH treatment. The first set of double labels (bottom arrow) was acquired before PTH treatment, and the second set of double labels (top arrow) was acquired after 3 months of PTH treatment.

Figure 5

A and B, Expression of osteoblast differentiation markers in sorted osteogenic cells. OCN⁺/CD34⁺ cells and OCN⁺/CD34⁻ cell populations were isolated. The expression of osteoblast differentiation marker genes was assessed by real-time PCR. Bars indicate means of duplicate determinations. C, Expression of osteoblast differentiation markers in cultured OCN⁺/CD34⁻ cells. Sorted OCN⁺/CD34⁻ cells were cultured in growth medium for 3 wk followed by either osteogenic differentiation media (black bars) or vehicle (gray bars) for an additional 3 wk. The expression of osteoblast differentiation marker genes was assessed by real-time PCR. *, P < 0.05. D and E, Alizarin red staining in cultured OCN⁺/CD34⁻ cells. Sorted OCN⁺/CD34⁻ cells were cultured in growth medium for 3 wk followed by either osteogenic differentiation media (D) or vehicle (E) for an additional 3 wk. Panel D shows positive staining as well as the presence of nodules.