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...

Experiments were carried out to determine whether the doses of the amino bisphosphonate ibandronate that inhibit bone resorption inhibit soft tissue calcification and death in rats treated with a toxic dose of vitamin D. These studies were prompted by the recent discovery that ibandronate doses that inhibit bone resorption potently inhibit artery calcification induced by treatment with the vitamin K antagonist warfarin. All 16 rats treated with the toxic dose of vitamin D (12.5 mg cholecalciferol x kg(-1)) died by d 6 after the first vitamin D injection (median survival: 4.5 d), whereas the 12 rats treated with vitamin D plus ibandronate (0.25 mg x kg(-1) x d(-1)) were alive and in good health at d 10. Rats treated with vitamin D alone and examined at d 4 had extensive Alizarin red staining for calcification in the aorta, the carotid, hepatic, mesenteric, renal and femoral arteries, kidneys and lungs, whereas rats treated with vitamin D plus ibandronate had no evidence for calcification at any of these tissues when examined at d 7 and 10. Ibandronate treatment also inhibited the dramatic increase in the levels of calcium and phosphate seen in the abdominal aorta, kidneys, lungs and trachea of the vitamin D-treated rats (P < 0.001). Serum calcium levels were, however, not different in rats treated with vitamin D alone (3.4 +/- 0.2 mmol x L(-1)) and in rats treated with vitamin D plus ibandronate (3.5 +/- 0.2 mmol x L(-1)). Treatment with vitamin D alone increased levels of matrix Gla protein, an inhibitor of soft tissue calcification, in the arteries, kidneys, lungs and trachea by 10- to 100-fold, and ibandronate treatment prevented this increase. The importance of these studies in the rat model is that they identify a class of drugs in current clinical use that can be used to treat patients with vitamin D toxicity and that they identify the dose of the drug that is predicted to be effective, namely the dose that inhibits bone resorption. Because there is no other known treatment for vitamin D toxicity, there would seem to be good reason to try bisphosphonates such as ibandronate in future studies aimed at treating patients who have been exposed to toxic levels of vitamin D.

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 ...