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 (