Endostatin inhibits VEGF-A induced osteoclastic bone resorption in vitro

Abstract

Background: Endostatin is a C-terminal fragment of collagen XVIII which is a component of basement membranes with the structural properties of both collagens and proteoglycans. Endostatin has a major role in angiogenesis which is intimately associated with bone development and remodeling. Signaling between the endothelial cells and the bone cells, for example, may have a role in recruitment of osteoclastic precursor cells. Our study aims at exploring a possibility that endostatin, either as a part of basement membrane or as a soluble molecule, may control osteoclastogenesis and osteoclastic bone resorption in vitro.

Methods: Rat pit formation assay was employed in order to examine the effect of endostatin alone or in combination with vascular endothelial growth factor-A (VEGF-A) on bone resorption in vitro. Effect of these agents on osteoclast differentiation in vitro was also tested. Osteoclastogenesis and the number of osteoclasts were followed by tartrate resistant acid phosphatase (TRACP) staining and resorption was evaluated by measuring the area of excavated pits.

Results: Endostatin inhibited the VEGF-A stimulated osteoclastic bone resorption, whereas endostatin alone had no effect on the basal resorption level in the absence of VEGF-A. In addition, endostatin could inhibit osteoclast differentiation in vitro independent of VEGF-A.

Conclusion: Our in vitro data indicate that collagen XVIII/endostatin can suppress VEGF-A induced osteoclastic bone resorption to the basal level. Osteoclastogenesis is also inhibited by endostatin. The regulatory effect of endostatin, however, is not critical since endostatin alone does not modify the basal bone resorption.

Figure 1

Number of TRACP+ multinuclear cells and the activity of bone resorption in pit assay. Rat bone cells were cultured for 48 hours with no added substances (CONTROL), vascular endothelial growth factor A (VEGF ng/ml), endostatin (ENDO μg/ml) or both VEGF-A and endostatin (VEGF ng/ml +ENDO μg/ml). (A) Survival rate of osteoclasts after different treatments was measured by counting the numbers of TRACP-positive cells with two or more nuclei indicating osteoclasts. No significant differences in the number of osteoclasts could be seen. (B) Osteoclastic bone resorption could be visualized with WGA-lectin stain and total resorbed areas from the different cultures were measured. VEGF-A treatment induced a clear stimulus, whereas endostatin caused no effect on the resorbed area. Addition of both VEGF-A and endostatin caused a statistically significant decrease in the total resorbed area when compared to the VEGF-A-induced resorption. (VEGF ng/ml, ENDO μg/ml)

Figure 2

Typical appearance of the bone slices in the pit assay. Rat bone cells were cultured on bovine bone slices with different treatments. After 48 hours, cells were wiped off and resorbed bone area was visualized with image analyzer system. Bone slices after (A) Control (B) VEGF-A 10 ng/ml (C) VEGF-A 50 ng/ml (D) VEGF-A 100 ng/ml (E) Endostatin 0.02 μg/ml (F) Endostatin 0.2 μg/ml (G) Endostatin 2 μg/ml (H) VEGF-A 10 ng/ml and Endostatin 0.02 μg/ml (I) VEGF-A 50 ng/ml and Endostatin 0.2 μg/ml (J) VEGF-A 100 ng/ml and Endostatin 2 μg/ml treatments. Scale bar 100 μm.

Figure 3

The number of TRACP+ multinuclear cells. Mouse (C57BL/6, 8–12 weeks) bone marrow cells were isolated from mouse long bones and seeded in 24-well plates on bovine bone slices. Cells on the bone slices were cultured in four groups: control, VEGF (100 ng/ml) and ENDO (2 μg/ml) and VEGF + ENDO respectively. The control group was cultured in α-MEM with 10% FCS containing 30 ng/ml RANKL and 10 ng/ml M-CSF for 7 days, after which the cultures were stopped by fixing the cells with 3% paraformaldehyde (PFA)/2% sucrose in PBS.