Protein engineering of cell surfaces is a potentially powerful technology through which the surface protein composition of cells can be manipulated without gene transfer. This technology exploits the fact that proteins that are anchored by glycoinositol phospholipids (GPIs), when purified and added to cells in vitro, incorporate into their surface membranes and are fully functional. By substituting 3'-mRNA end sequence of naturally GPI-anchored proteins (i.e., a sequence that contains the signals that direct GPI anchoring) for endogenous 3'-mRNA end sequence, virtually any protein of interest can be expressed as a GPI-anchored derivative. The GPI-anchored product then can be purified from transfectants and the purified protein used to "paint" any target cell. Such protein engineering or "painting" of the cell surface offers several advantages over conventional gene transfer. Among these advantages are that 1) GPI-anchored proteins can be painted onto cells that are difficult to transfect, 2) cells can be altered immediately without previous culturing, 3) the amount of protein added to the surface can be precisely controlled, and 4) multiple GPI-anchored proteins can be sequentially or concurrently inserted into the same cells. Emerging applications for the technology include its use for the analysis of complex cell-surface interactions, the engineering of antigen presenting cells, the development of cancer vaccines, and possibly the protection against graft rejection.

Hypercholesterolemia is a major risk factor for atherosclerosis. It also is associated with platelet hyperactivity, which increases morbidity and mortality from cardiovascular disease. However, the mechanisms by which hypercholesterolemia produces a procoagulant state remain undefined. Atherosclerosis is associated with accumulation of oxidized lipoproteins within atherosclerotic lesions. Small quantities of oxidized lipoproteins are also present in the circulation of patients with coronary artery disease. We therefore hypothesized that hypercholesterolemia leads to elevated levels of oxidized LDL (oxLDL) in plasma and that this induces expression of the procoagulant protein tissue factor (TF) in monocytes. In support of this hypothesis, we report here that oxLDL induced TF expression in human monocytic cells and monocytes. In addition, patients with familial hypercholesterolemia had elevated levels of plasma microparticle (MP) TF activity. Furthermore, a high-fat diet induced a time-dependent increase in plasma MP TF activity and activation of coagulation in both LDL receptor-deficient mice and African green monkeys. Genetic deficiency of TF in bone marrow cells reduced coagulation in hypercholesterolemic mice, consistent with a major role for monocyte-derived TF in the activation of coagulation. Similarly, a deficiency of either TLR4 or TLR6 reduced levels of MP TF activity. Simvastatin treatment of hypercholesterolemic mice and monkeys reduced oxLDL, monocyte TF expression, MP TF activity, activation of coagulation, and inflammation, without affecting total cholesterol levels. Our results suggest that the prothrombotic state associated with hypercholesterolemia is caused by oxLDL-mediated induction of TF expression in monocytes via engagement of a TLR4/TLR6 complex.

No abstract

Hepatic repair is directed chiefly by the proliferation of resident mature epithelial cells. Further if predominant injury is to cholangiocytes, the hepatocytes can transdifferentiate to cholangiocytes to assist in the repair and vice versa as shown by various fate-tracing studies. However, the molecular bases of reprograming remain elusive. Using two models of biliary injury where repair occurs via cholangiocyte proliferation and hepatocyte transdifferentiation to cholangiocytes, we identify an important role of Wnt signaling. First we identify upregulation of specific Wnt proteins in the cholangiocytes. Next, using conditional knockouts of Wntless and Wnt co-receptors LRP5/6, transgenic mice expressing stable β-catenin, and in vitro studies, we show a role of Wnt signaling through β-catenin in hepatocyte to biliary transdifferentiation. Lastly, we show that specific Wnts regulate cholangiocyte proliferation but in a β-catenin-independent manner. Conclusion: Wnt signaling regulates hepatobiliary repair after cholestatic injury in both β-catenin dependent and independent manners.