Abstract-High doses of warfarin cause focal calcification of the elastic lamellae in the media of major arteries and in aortic heart valves in the rat. Aortic calcification was first seen after 2 weeks of warfarin treatment and progressively increased in density at 3, 4, and 5 weeks of treatment. By 5 weeks, the highly focal calcification of major arteries could be seen on radiographs and by visual inspection of the artery. The calcification of arteries induced by warfarin is similar to that seen in the matrix Gla protein (MGP)-deficient mouse, which suggests that warfarin induces artery calcification by inhibiting ␥-carboxylation of MGP and thereby inactivating the putative calcification-inhibitory activity of the protein. Warfarin treatment markedly increased the levels of MGP mRNA and protein in calcifying arteries and decreased the level of MGP in serum. Warfarin treatment did not affect bone growth, overall weight gain, or serum calcium and phosphorus levels, and, because of the concurrent administration of vitamin K, prothrombin times and hematocrits were normal. The results indicate that the improved warfarin plus vitamin K treatment protocol developed in this study should provide a useful model to investigate the role of MGP in preventing calcification of arteries and heart valves.

1447 on chicken bone a presumptive Gla fraction was isolated by ion exchange chromatography from alkaline hydrolysates of the chicken bone and identified as Gla on the basis of both its conversion to glutamic acid upon heating in acid and its coelution with a similar putative Gla fraction in alkaline hydrolysates of prothrombin (7). In the present study we have used Gla synthesized by the procedure of Morris et al. (9) to show further that the mass spectra of the presumptive Gla from bovine bone and synthetic Gla are identical. Studies with synthetic Gla have also enabled us to show that Gla is indeed completely stable under the conditions of alkaline protein hydrolysis, and to establish its ninhydrin color factor for quantitative estimation of Gla content in proteins.In the present work we report the chemical composition of the bovine Gla protein and the sequence of its first 15 residues, which establish that the bovine Gla protein is not a fragment of the bovine blood clotting factors. We also present evidence of a similar Gla-containing protein in most other bovine calcified tissues and in a variety of other vertebrates. The low molecular weight and high electrophoretic mobility of the main Gla protein in chicken bone (7) indicate that it is probably another member of this class of proteins. We find that the bovine Gla protein binds strongly to the mineral phase of bone, with an average stoichiometry of one Gla protein per crystal, and that the bovine Gla protein is a potent inhibitor of hydroxyapatite crystallization. MATERIALS AND METHODSCortical bone was obtained from the central section of the femur of freshly slaughtered calves or cows. Bone samples were freed of marrow and connective tissue, ground to a particle size that passed through a 210-,gm sieve, and washed with several changes of water for 24 hr at 4°. The bone was then dialyzed against several changes of 0.5 M EDTA, pH 8, at 40 for 8-10 days. The soluble fraction inside the dialysis sack was collected by centrifugation, dialyzed exhaustively against 5 mM NH4HCO3, and lyophilized. After gel filtration on Sephadex G-100 (Fig. 1), peak fractions containing the Gla protein were lyophilized and then chromatographed on a 2 X 50 cm column of DEAE-Sephadex A25 at 25°with a linear gradient in 0.1 M Tris-HCI, pH 8.0, from 0 to 0.75 M NaCl. The procedure for the isolation of Gla protein from swordfish and human bone and bovine dentine was altered only in the use of 50 mM rather than 5 mM NH4HCO3 to suppress Gla protein precipitation. Hydroxyapatite crystals were made by mixing calcium chloride and sodium phosphate solutions at 5 mM concentrations, pH 7.4, and 250. Hydroxyapatite also was purchased from Clarkson Chemical Co. Amorphous calcium phosphate was prepared by mixing saturated solutions of calcium chloride and sodium phosphate at pH 7.4 and 250.

Primary pneumonic plague is transmitted easily, progresses rapidly, and causes high mortality, but the mechanisms by which Yersinia pestis overwhelms the lungs are largely unknown. We show that the plasminogen activator Pla is essential for Y. pestis to cause primary pneumonic plague but is less important for dissemination during pneumonic plague than during bubonic plague. Experiments manipulating its temporal expression showed that Pla allows Y. pestis to replicate rapidly in the airways, causing a lethal fulminant pneumonia; if unexpressed, inflammation is aborted, and lung repair is activated. Inhibition of Pla expression prolonged the survival of animals with the disease, offering a therapeutic option to extend the period during which antibiotics are effective.

YKL-40, a member of the ''mammalian chitinase -like

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...