Recombinant TFPI-2 enhances macrophage apoptosis through upregulation of Fas/FasL.

Tissue factor pathway inhibitor-2 (TFPI-2) is a Kunitz-type serine proteinase inhibitor with inhibitory activity toward activated factor XI, plasma kallikrein, plasmin, certain matrix metalloproteinases, and the tissue factor-activated factor VII complex. In addition, TFPI-2 has other functions such as promoting cell migration and inducing apoptosis. In the present study, we investigated if TFPI-2 induced apoptosis in cultured U937-derived macrophages and the possible signal pathways that involved in the apoptotic process. Apoptotic DNA fragment detection and caspase-3,9 activity measurements indicated that rTFPI-2 promoted U937-derived macrophage apoptosis. Hoechst 33342 assay and flow cytometry further showed that rTFPI-2 induced apoptosis in cultured macrophages in a dose-dependent manner. Because death receptors of the TNF family such as Fas are the best-understood death pathways that recruit Fas-associated death domain (FADD) and procaspase-8 to the receptor in macrophages, we investigated the expression of Fas and its ligand (FasL) and downstream signal caspase-8 by Western blot analysis. The results indicated that the process of apoptosis triggered by rTFPI-2 was, at least in part, actively conducted by U937-derived macrophages possibly through Fas/FasL signal pathway. In brief, rTFPI-2 may have the potential usefulness in inducing macrophages apoptosis, which suggest TFPI-2 might have antiatherogenic effects.

Peptide corresponding to the C terminus of tissue factor pathway inhibitor inhibits mesangial cell proliferation and activation in vivo.

Mesangial cells (MsCs) are one of the resident cell types in the glomerulus and are important with respect to its function and structure. The activation and proliferation of MsCs occur in several types of glomerulonephritis, particularly proliferative glomerulonephritis, producing a series of protein factors and matrix components that impair the normal structure and function of the glomerulus. To inhibit proliferation or induction of apoptosis is considered to be one mechanism that can be used to treat these diseases. In previous studies, we found that the tissue factor pathway inhibitor (TFPI) induces the apoptosis of cultured rat MsCs. Here, we expressed a series of TFPI fragments as fusion proteins to maltose binding protein (MBP-TFPI(162-188), MBP-TFPI(187-241), MBP-TFPI(240-276), MBP-TFPI(162-241), MBP-TFPI(187-276) and MBP-TFPI(162-276)) and applied them to cultured rat mesangial cells. The C terminus of TFPI, a peptide corresponding to residues 240-276 of TFPI, was confirmed to induce apoptosis of MsCs in vitro. To observe the effect of this peptide on MsCs in vivo, we performed intramuscular gene transfer treatment on a rat model of proliferative glomerulonephritis with a plasmid containing the gene for the C terminus of TFPI. This revealed that the C terminus of TFPI exhibited suppressive effects on the activation and proliferation of MsCs and, thereby, improved renal function. Our data indicate that the C terminus of TFPI could be used in the treatment of proliferative glomerulonephritis.