Abstract-The present experiments were carried out to test the hypothesis that arterial calcification is linked to bone resorption by determining whether the selective inhibition of bone resorption with osteoprotegerin will inhibit arterial calcification. In the first test, arterial calcification was induced by treating 22-day-old male rats with warfarin, a procedure that inhibits the ␥-carboxylation of matrix Gla protein and causes extensive calcification of the arterial media.Compared with rats treated for 1 week with warfarin alone, rats treated with warfarin plus osteoprotegerin at a dose of 1 mg/kg per day had dramatically reduced alizarin red staining for calcification in the aorta and in the carotid, hepatic, mesenteric, renal, and femoral arteries, and they had 90% lower levels of calcium and phosphate in the abdominal aorta (PϽ0.001) and in tracheal ring cartilage (PϽ0.01). More rapid arterial calcification was induced by treating 49-day-old male rats with toxic doses of vitamin D. Treatment for 96 hours with vitamin D caused widespread alizarin red staining for calcification in the aorta and the femoral, mesenteric, hepatic, renal, and carotid arteries, and osteoprotegerin completely prevented calcification in each of these arteries and reduced the levels of calcium and phosphate in the abdominal aorta to control levels (PϽ0.001). Treatment with vitamin D also caused extensive calcification in the lungs, trachea, kidneys, stomach, and small intestine, and treatment with osteoprotegerin reduced or prevented calcification in each of these sites. Measurement of serum levels of cross-linked N-teleopeptides showed that osteoprotegerin dramatically reduced bone resorption activity in each of these experiments (PϽ0.001). Therefore, we conclude that doses of osteoprotegerin that inhibit bone resorption are able to potently inhibit the calcification of arteries that is induced by warfarin treatment and by vitamin D treatment. 1,2 to explain the association between the increased bone resorption and increased arterial calcification that has been seen in the vitamin D-treated rat, 1 in the osteoprotegerin-deficient mouse, 3 and in patients with postmenopausal osteoporosis. 4 -12 One prediction of the hypothesis that arterial calcification is linked to bone resorption is that inhibitors of bone resorption should inhibit arterial calcification. 2 In previous studies, we have shown that arterial calcification induced by treatment with warfarin and by treatment with high doses of vitamin D is indeed inhibited by 2 potent inhibitors of bone resorption, the amino bisphosphonates alendronate and ibandronate, at doses of these drugs known to inhibit bone resorption in the rat. 2,13 In the present investigations, we have determined the effect of another potent inhibitor of bone resorption, osteoprotegerin, on arterial calcification by using subcutaneous doses of this protein that have been shown to inhibit bone resorption in the rat.Osteoprotegerin is a secreted protein of the tumor necrosis factor family, which regulat...

The present studies show for the first time that demineralized bone re-calcifies rapidly when incubated at 37°C in rat serum: re-calcification can be demonstrated by Alizarin Red and von Kossa stains, by depletion of serum calcium, and by uptake of calcium and phosphate by bone matrix. Re-calcification is specific for the type I collagen matrix structures that were calcified in the original bone, with no evidence for calcification in periosteum or cartilage. Re-calcification ceases when the amount of calcium and phosphate introduced into the matrix is comparable to that present in the original bone prior to demineralization, and the re-calcified bone is palpably hard. Re-calcified bone mineral is comparable to the original bone mineral in calcium to phosphate ratio and in Fourier transform infrared and x-ray diffraction spectra. The serum activity responsible for re-calcification is sufficiently potent that the addition of only 1.5% serum to Dulbecco's modified Eagle's medium causes bone re-calcification. This putative serum calcification factor has an apparent molecular mass of 55-150 kDa and is inactivated by trypsin or chymotrypsin. The serum calcification factor must act on bone for 12 h before re-calcification can be detected by Alizarin Red or von Kossa staining and before the subsequent growth of calcification will occur in the absence of serum. The speed, matrix-type specificity, and extent of the seruminduced re-calcification of demineralized bone suggest that the serum calcification factor identified in these studies may participate in the normal calcification of bone.This study is a continuation of our investigations into the mechanisms that initiate the normal calcification of bone and the abnormal calcification of arteries and into the mechanisms that inhibit these calcifications. We recently proposed the hypothesis that artery calcification is linked to bone resorption (1, 2) to explain the association between increased bone resorption and increased artery calcification that has been seen in the vitamin D-treated rat (1), in the osteoprotegerin-deficient mouse (3), and in patients with postmenopausal osteoporosis (see Ref. 2 for references). One prediction of the hypothesis that artery calcification is linked to bone resorption is that inhibitors of bone resorption should inhibit artery calcification (2). In previous studies we tested this prediction using three different types of bone resorption inhibitors, each with an entirely different mode of action on the osteoclast, the amino bisphosphonates alendronate and ibandronate (2), the cytokine osteoprotegerin (4), and the V-H ϩ -ATPase inhibitor SB 242784 (5). Each bone resorption inhibitor potently inhibited artery calcification.Ibandronate, osteoprotegerin, and SB 242784 are each highly specific inhibitors of the osteoclast at the concentrations used in these studies and have no known actions on vascular cells. Their ability to potently inhibit vascular calcification is therefore hard to reconcile with the hypothesis that the process is initiated...

Abstract-The present experiments were carried out to further test the hypothesis that arterial calcification is linked to bone resorption by determining whether the selective inhibition of bone resorption with SB 242784, a specific inhibitor of the osteoclastic V-H ϩ -ATPase, will inhibit arterial calcification. Treatment for 96 hours with toxic doses of vitamin D caused widespread calcification in the aorta and in the femoral, mesenteric, hepatic, renal, and carotid arteries, and treatment with SB 242784 completely prevented the vitamin D-induced calcification of each of these arteries at a dose of 40 mg/kg per day and significantly reduced calcification at a dose of 10 mg/kg per day. Treatment with vitamin D also caused extensive calcification in the lungs, tracheal cartilage, and kidneys, and treatment with SB 242784 prevented or reduced calcification at each of these sites. Measurement of serum levels of cross-linked N-telopeptides, a specific measure of bone resorption activity, showed that treatment with vitamin D alone produced the expected 2.4-fold increase in bone resorption activity and that concurrent treatment with the 40-mg dose of SB 242784 reduced bone resorption activity to below control levels. With the inclusion of the present results, there are now three types of bone resorption inhibitors (each with an entirely different mode of action on the osteoclast) that share the ability to potently inhibit arterial calcification in the rat, the V-H ϩ -ATPase inhibitor SB 242784, the cytokine osteoprotegerin, and the amino bisphosphonates alendronate and ibandronate. (Circ Res. 2002;91:547-552.)e recently proposed the hypothesis that arterial calcification is linked to bone resorption 1,2 to explain the association between increased bone resorption and increased arterial calcification that has been seen in the vitamin D-treated rat, 1 in the osteoprotegerin-deficient mouse, 3 and in patients with postmenopausal osteoporosis. 4 -12 One prediction of the hypothesis that arterial calcification is linked to bone resorption is that inhibitors of bone resorption should inhibit arterial calcification. 2 In a previous study, we showed that arterial calcification induced by treatment with warfarin and by treatment with high doses of vitamin D is indeed inhibited by two potent inhibitors of bone resorption, the amino bisphosphonates alendronate and ibandronate, at doses of these drugs known to inhibit bone resorption in the rat. 2,13 In a subsequent investigation, we showed that another potent inhibitor of bone resorption, the cytokine osteoprotegerin, also inhibited arterial calcification at subcutaneous doses of osteoprotegerin that have been shown to inhibit bone resorption in the rat. 14 In the present investigation, we have further tested the hypothesis that arterial calcification is linked to bone resorption by determining the effect of a potent inhibitor of bone resorption with an entirely different mode of action, the small molecule SB 242784, on arterial calcification in the rat.SB 242784 inhibits bone ...