Osteoprotegerin and RANKL differentially regulate angiogenesis and endothelial cell function

Abstract

Osteoprotegerin (OPG) a soluble tumor necrosis factor receptor family molecule protects endothelial cells from apoptosis in vitro and promotes neovascularization in vivo. In this study, we assessed the role of OPG and its ligands, receptor activator of nuclear factor-kappaB ligand (RANKL) and tumor necrosis factor-related apoptosis inducing ligand (TRAIL), in microvessel formation using the rat aortic ring model of angiogenesis. OPG was found to promote a twofold increase in angiogenic sprouting in the aortic ring model, and this effect was inhibited by pre-incubation with a fivefold molar excess of either RANKL or TRAIL. While TRAIL had no effect upon angiogenesis on its own, RANKL was found to potently inhibit basal and vascular endothelial growth factor-induced angiogenesis. OPG increased the rate of endothelial cell proliferation in sprouting microvessels; in contrast, RANKL inhibited proliferation. RANKL was found to induce endothelial apoptosis at days 6, 7, and 10 in the aortic ring model and after incubation with human umbilical vein endothelial cells (HUVECs). Signaling studies showed that OPG induced ERK1/2 and Akt phosphorylation in HUVECs while RANKL had no effect. Our results indicate that OPG is a positive regulator of microvessel formation, while RANKL is an angiogenic inhibitor due to effects on regulation of endothelial cell proliferation, apoptosis, and signaling.

Figure 1.

RANKL and RANK are expressed in rat aorta and isolated endothelial cells. OPG is expressed in rat aorta (A) RT-PCR for RANKL and RANK in rat tissues. 1 = Thymus; 2 = Spleen; 3 = Aorta; 4 = Negative Control Aorta –RT. (B) Western Blot for RANKL and RANK in HUVECs. Arrows indicate bands corresponding to RANKL membrane bound form (40kDa), soluble RANKL form (30kDa), and RANK (80kDa). (C) Western Blot for OPG in rat aorta. Arrow indicate band corresponding to OPG dimer (110kDa).

Figure 2.

OPG promotes angiogenesis in vitro. (A) Vessel growth curve for OPG and untreated control rings (Mean +/− SEM, n = 3). Microvessels sprouts were counted at the indicated times. At the same timepoints fresh media with or without treatment was added. (B) Representative 2x light microscopy images from day 8 in culture. Bar, 500 µm.

Figure 3.

Both TRAIL and RANKL block OPG-induced angiogenesis. Treatments were added to aortic ring cultures every other day. Microvessels sprouts were counted at the same timepoints. Day 7 counts that correspond to the maximum number of microvessels are shown. (A) Day 7 vessel scores for OPG and OPG/TRAIL combination treated rings (Mean +/− SEM, n = 3). (B) Day 7 vessel scores for OPG and OPG/RANKL combination treated rings (Mean +/− SEM, n = 3).

Figure 4.

RANKL inhibits angiogenesis. Microvessels sprouts were counted at the indicated times. At the same timepoints fresh media with or without treatment was added. (A) Vessel scores for RANKL treated rings (Mean +/− SEM, n = 3). (B) Representative 2x light microscopy images from day 7 in culture. Bar, 500 µm. (C) Vessel scores for VEGF and VEGF/RANKL combination treated rings (Mean +/− SEM, n = 4).

Figure 5.

(A) Quantification of endothelial specific apoptosis in aortic rings (Mean +/− SEM, n = 3). Microvessels sprouts were counted at the indicated times. At the same timepoints fresh media with or without treatment was added. Aortic ring cultures were fixed and stained at the indicated timepoints. (B) Sample fluorescent images from day 6 aortic rings. White arrows indicate apoptotic endothelial cells. Bar, 100 µm. (C) Quantification of apoptosis in HUVECs (Mean +/− SEM, n=3). HUVECs were synchronized in serum free condition for 24 hours and then treated with the indicated factors for additional 24 hours before fixing and staining for apoptotic cells determination.

Figure 6.

(A) Quantification of endothelial proliferation at day 6 in culture (Mean +/− SEM, n = 6). Microvessels sprouts were counted at the indicated times. At the same timepoints fresh media with or without treatment was added. Aortic ring cultures were fixed and stained after 6 days in culture. (B) Vessel scores for cultures in 6A (Mean +/− SEM, n = 6). (C) Sample overlay images from day 6 aortic rings. Proliferating endothelial cells are indicated with white arrows. Bar, 100 µm.

Figure 7.

OPG activates ERK1/2 and Akt signaling pathways in HUVECs (A) Western Blots from HUVECs treated with 5 nM OPG or vehicle control for 5, 15 and 30 minutes. (B) Western Blots from HUVECs treated with 50 nM RANKL or vehicle control for 5, 15 and 30 minutes. (C) Quantification of ERK1/2 phosphorylation. (D) Quantification of Akt phosphorylation. Normalized values from three independent experiments were averaged and the standard deviation calculated.