Abstract:The causes of arterial calci®cation are beginning to be elucidated. Macrophages, mast cells, and smooth muscle cells are the primary cells implicated in this process. The roles of a variety of bone-related proteins including bone morphogenetic protein-2 (BMP-2), matrix Gla protein (MGP), osteoprotegerin (OPG), osteopontin, and osteonectin in regulating arterial calci®cation are reviewed. Animals lacking MGP, OPG, smad6, carbonic anhydrase isoenzyme II, ®brillin-1, and klotho gene product develop varying extents of arterial calci®cation. Hyperlipidemia, vitamin D, nicotine, and warfarin, alone or in various combinations, produce arterial calci®cation in animal models. MGP has recently been discovered to be an inhibitor of bone morphogenetic protein-2, the principal osteogenic growth factor. Many of the forces that induce arterial calci®cation may act by disrupting the essential post-translational modi®cation of MGP, allowing BMP-2 to induce mineralization. MGP requires gamma-carboxylation before it is functional, and this process uses vitamin K as an essential cofactor. Vitamin K de®ciency, drugs that act as vitamin K antagonists, and oxidant stress are forces that could prevent the formation of GLA residues on MGP. The potential role of arterial apoptosis in calci®cation is discussed. Potential therapeutic options to limit the rate of arterial calci®cation are summarized.

Background: Erythrocytes contribute to nitrite-mediated NO signaling, but the mechanism is unclear. Results: Deoxyhemoglobin accounts for virtually all NO made from nitrite by erythrocytes with no contributions from other proposed pathways. Conclusion: Deoxyhemoglobin is the primary erythrocytic nitrite reductase operating under physiological conditions. Significance: Reduction by deoxyhemoglobin accounts for nitrite-mediated NO signaling in blood mediating vessel tone and platelet function.

The vitamin K-dependent ␥-carboxylation system in the endoplasmic reticulum membrane responsible for ␥-carboxyglutamic acid modification of vitamin K-dependent proteins includes ␥-carboxylase and vitamin K 2,3-epoxide reductase (VKOR). An understanding of the mechanism by which this system works at the molecular level has been hampered by the difficulty of identifying VKOR involved in warfarin sensitive reduction of vitamin K 2,3-epoxide to reduced vitamin K 1 H 2 , the ␥-carboxylase cofactor. Identification and cloning of VKORC1, a proposed subunit of a larger VKOR enzyme complex, have provided opportunities for new experimental approaches aimed at understanding the vitamin K-dependent ␥-carboxylation system. In this work we have engineered stably transfected baby hamster kidney cells containing ␥-carboxylase and VKORC1 cDNA constructs, respectively, and stably double transfected cells with the ␥-carboxylase and the VKORC1 cDNA constructs in a bicistronic vector. All engineered cells showed increased activities of the enzymes encoded by the cDNAs. However increased activity of the ␥-carboxylation system, where VKOR provides the reduced vitamin K 1 H 2 cofactor, was measured only in cells transfected with VKORC1 and the double transfected cells. The results show that VKOR is the rate-limiting step in the ␥-carboxylation system and demonstrate successful engineering of cells containing a recombinant vitamin K-dependent ␥-carboxylation system with enhanced capacity for ␥-carboxyglutamic acid modification.

Some recombinant vitamin K-dependent blood coagulation factors (factors VII, IX, and protein C) have become valuable pharmaceuticals in the treatment of bleeding complications and sepsis. Because of their vitamin K-dependent post-translational modification, their synthesis by eukaryotic cells is essential. The eukaryotic cell harbors a vitamin K-dependent ␥-carboxylation system that converts the proteins to ␥-carboxyglutamic acid-containing proteins. However, the system in eukaryotic cells has limited capacity, and cell lines overexpressing vitamin K-dependent clotting factors produce only a fraction of the recombinant proteins as fully ␥-carboxylated, physiologically competent proteins. In this work we have used recombinant human factor IX (r-hFIX)-producing baby hamster kidney (BHK) cells, engineered to stably overexpress various components of the ␥-carboxylation system of the cell, to determine whether increased production of functional r-hFIX can be accomplished. All BHK cell lines secreted r-hFIX into serum-free medium. Overexpression of ␥-carboxylase is shown to inhibit production of functional r-hFIX. On the other hand, cells overexpressing VKORC1, the reduced vitamin K cofactor-producing enzyme of the vitamin Kdependent ␥-carboxylation system, produced 2.9-fold more functional r-hFIX than control BHK cells. The data are consistent with the notion that VKORC1 is the ratelimiting step in the system and is a key regulatory protein in synthesis of active vitamin K-dependent proteins. The data suggest that overexpression of VKORC1 can be utilized for increased cellular production of recombinant vitamin K-dependent proteins.

Abstract-Hepatocyte growth factor (scatter factor) is an angiogenic growth factor that binds to its cellular transmembrane receptor, c-met. Both HGF and c-met are expressed by vascular smooth muscle and endothelial cells, where HGF may exert autocrine and paracrine effects. We have found that human aortic smooth muscle cells (

Matrix ␥-carboxyglutamic acid protein (MGP) is a member of the vitamin K-dependent protein family with unique structural and physical properties. MGP has been shown to be an inhibitor of arterial wall and cartilage calcification. One inhibitory mechanism is thought to be binding of bone morphogenetic protein-2. Binding has been shown to be dependent upon the vitamin K-dependent ␥-carboxylation modification of MGP. Since MGP is an insoluble matrix protein, this work has focused on intracellular processing and transport of MGP to become an extracellular binding protein for bone morphogenetic protein-2. Human vascular smooth muscle cells (VSMCs) were infected with an adenovirus carrying the MGP construct, which produced non-␥-carboxylated MGP and fully ␥-carboxylated MGP. Both forms of MGP were found in the cytosolic and microsomal fractions obtained from the cells by differential centrifugation. The crude microsomal fraction was shown to contain an additional, more acidic Ser-phosphorylated form of MGP believed to be the product of Golgi casein kinase. The data suggest that phosphorylation of MGP dictates different transport routes for MGP in VSMCs. A proteomic approach failed to identify a larger soluble precursor of MGP or an intracellular carrier protein for MGP. Evidence is presented for a receptormediated uptake mechanism for fetuin by cultured human VSMCs. Fetuin, shown by mass spectrometry not to contain MGP, was found to be recognized by anti-MGP antibodies. Fetuin uptake and secretion by proliferating and differentiating cells at sites of calcification in the arterial wall may represent an additional protective mechanism against arterial calcification.

The discovery of novel globins in diverse organisms has stimulated intense interest in their evolved function, beyond oxygen binding. Globin X (GbX) is a protein found in fish, amphibians, and reptiles that diverged from a common ancestor of mammalian hemoglobins and myoglobins. Like mammalian neuroglobin, GbX was first designated as a neuronal globin in fish and exhibits six-coordinate heme geometry, suggesting a role in intracellular electron transfer reactions rather than oxygen binding. Here, we report that GbX to our knowledge is the first six-coordinate globin and the first globin protein apart from hemoglobin, found in vertebrate RBCs. GbX is present in fish erythrocytes and exhibits a nitrite reduction rate up to 200-fold faster than human hemoglobin and up to 50-fold higher than neuroglobin or cytoglobin. Deoxygenated GbX reduces nitrite to form nitric oxide (NO) and potently inhibits platelet activation in vitro, to a greater extent than hemoglobin. Fish RBCs also reduce nitrite to NO and inhibit platelet activation to a greater extent than human RBCs, whereas GbX knockdown inhibits this nitrite-dependent NO signaling. The description of a novel, sixcoordinate globin in RBCs with dominant electron transfer and nitrite reduction functionality provides new insights into the evolved signaling properties of ancestral heme-globins.nitrite | nitric oxide | blood | RBC | platelet

Background-Our objective was to determine whether abciximab, eptifibatide, or tirofiban inhibited ligand binding to ␣ v ␤ 3 integrins on human aortic smooth muscle cells (HASMCs) or human umbilical vein endothelial cells (HUVECs). Abciximab binds ␣ IIb ␤ 3 on platelets and ␣ v ␤ 3 on HUVECs with similar affinity, whereas eptifibatide and tirofiban are thought to be highly specific for ␣ IIb ␤ 3 . The conclusion that eptifibatide does not bind vascular ␣ v ␤ 3 integrins may be premature, however, because recent studies have demonstrated that the affinity of ␣ v ␤ 3 for various ligands, including antagonists, is subject to modulation. Methods and Results-Abciximab and 7E3, the anti-␤ 3 integrin monoclonal antibody from which abciximab was derived, bound ␣ v ␤ 3 on HASMCs in a specific and saturable manner and with an affinity similar to binding to ␣ IIb ␤ 3 on platelets. 7E3 and eptifibatide inhibited ␣ v ␤ 3 -mediated attachment of HASMCs to thrombospondin (TSP) and prothrombin but had no effect on ␣ v ␤ 5 -or ␤ 1 -mediated HASMC attachment to vitronectin-, collagen-, or fibronectin-coated or noncoated tissue culture plates. The inhibitory effect of eptifibatide was similar in magnitude and not additive to that of 7E3.