Abstract-Vascular calcification is associated with an increased risk of myocardial infarction; however, the mechanisms linking these 2 processes are unknown. Studies in macrophages have suggested that calcium phosphate crystals induce the release of proinflammatory cytokines; however, no studies have been performed on the effects of calcium phosphate crystals on vascular smooth muscle cell function. In the present study, we found that calcium phosphate crystals induced cell death in human aortic vascular smooth muscle cells with their potency depending on their size and composition. Calcium phosphate crystals of approximately 1 m or less in diameter caused rapid rises in intracellular calcium concentration, an effect that was inhibited by the lysosomal proton pump inhibitor, bafilomycin A1. Bafilomycin A1 also blocked vascular smooth muscle cell death suggesting that crystal dissolution in lysosomes leads to an increase in intracellular calcium levels and subsequent cell death. These studies give novel insights into the bioactivity of calcified deposits and suggest that small calcium phosphate crystals could destabilize atherosclerotic plaques by initiating inflammation and by causing vascular smooth muscle cell death. (Circ Res. 2008;103:e28-e34.)

The nef gene of human and simian immunodeficiency viruses is critical for AIDS pathogenesis. Its function in vivo is unknown, but in vitro natural isolates of Nef down-regulate expression of the cell surface CD4 molecule, a component of the T cell antigen receptor and the viral receptor, by accelerating its endocytosis. We have used chimeric proteins comprised of the natural HIV-1 NA7 Nef fused to a strongly fluorescing mutant of green fluorescent protein (GFP) to correlate Nef function with intracellular localization in human CD4-positive Jurkat T cells. The NA7-GFP fusion protein co-localizes with components of the clathrin coat, including clathrin and the β-subunit of the AP-2 adaptor protein complex, at discrete locations that are consistent with the normal cellular distribution of clathrin coats at the plasma membrane. The NA7-GFP protein is also found in the perinuclear region of the cell, which is likely to reflect the Golgi apparatus. Evidence from a CD4-negative fibroblast cell line indicates that co-localization of NA7-GFP with components of the clathrin coat does not require expression of the CD4 molecule. Analysis of a large panel of chimeric molecules containing mutant Nef moieties demonstrated that the N-terminal membrane targeting signal cooperates with additional element(s) in the disordered loops in the Nef molecule to co-localize the Nef protein with AP-2 adaptor complexes at the cell margin. This localization of NA7-GFP correlates with, but is not sufficient for, down-regulation of surface CD4 and at least one additional function of Nef is required. In T cells co-expressing CD4 and NA7-GFP, CD4 at the cell surface is redistributed into a discrete pattern that co-localizes with that of NA7-GFP. Our observations place NA7-GFP in physical proximity to AP-2-containing clathrin coat at the plasma membrane and imply that Nef interacts, either directly or indirectly, with a component of the AP-2-containing coat at this location. This evidence supports a model whereby Nef recruits CD4 to the endocytic machinery via AP-2-containing clathrin coats at the plasma membrane.