ABSTRACT
Limiting the acute vascular damage associated with leukocyte infiltration is a central issue in solid organ transplantation. The family of chemotactic cytokines (chemokines) helps to regulate leukocyte recruitment. Systemic treatment with the chemokine ligand-5 (CCL5) based antagonist Met-RANTES has previously shown to suppress acute damage to transplanted kidneys by blocking effector cell recruitment. To address problems associated with systemic long-term administration of chemokine receptor antagonists, a chemokine based reagent was designed to be integrated into endothelial surfaces of the organ just before transplantation. Proteins anchored by glycosylphosphatidylinositol (GPI), when purified and added to cells, are efficiently incorporated into their cell surface membranes. A series of modifications were introduced into the CCL5 protein to generate a functional antagonist. These included the addition of an N-terminal methionine group, a mutation to render the protein a dimer and a GPI signal sequence for surface expression. The resultant protein was stably expressed in CHO cells, GPI anchorage was confirmed and the protein purified by FPLC. Exogenously administered Met-CCL5(dimer)-GPI was efficiently inserted into the membrane of microvascular endothelial cells. The reagent is being tested in murine models of renal transplantation. The effect on subsequent immune induced damage will be assessed.
Subject(s)
Chemokines, CC/chemistry , Endothelium, Vascular/drug effects , Glycosylphosphatidylinositols/metabolism , Receptors, Chemokine/antagonists & inhibitors , Transplantation, Homologous , Acute Disease/therapy , Animals , Base Sequence , CHO Cells , Cells, Cultured , Chemokine CCL5 , Cricetinae , Endothelium, Vascular/pathology , Humans , Kidney Transplantation , Mice , Molecular Sequence Data , MutationABSTRACT
The family of tissue inhibitors of metalloproteinases (TIMPs) exhibits diverse physiological/biological functions including the inhibition of active matrix metalloproteinases, regulation of proMMP activation, cell growth, and the modulation of angiogenesis. TIMP-1 is a secreted protein that can be detected on the cell surface through its interaction with surface proteins. The diverse biological functions of TIMP-1 are thought to lie, in part, in the kinetics of TIMP-1/MMP/surface protein interactions. Proteins anchored by glycoinositol phospholipids (GPIs), when purified and added to cells in vitro, are incorporated into their surface membranes. A GPI anchor was fused to TIMP-1 to generate a reagent that could be added directly to cell membranes and thus focus defined concentrations of TIMP-1 protein on any cell surface independent of protein-protein interaction. Unlike native TIMP-1, exogenously added GPI-anchored TIMP-1 protein effectively blocked release of MMP-2 and MMP-9 from osteosarcoma cells. TIMP-1-GPI was a more effective modulator of migration and proliferation than TIMP-1. While control hTIMP-1 protein did not significantly affect migration of primary microvascular endothelial cells at the concentrations tested, the GPI-anchored TIMP-1 protein showed a pronounced suppression of endothelial cell migration in response to bFGF. In addition, TIMP-1-GPI was more effective at inducing microvascular endothelial proliferation. In contrast, fibroblast proliferation was suppressed by the agent. Reagents based on this method should assist in the dissection of the protease cascades and activities involved in TIMP biology. Membrane-fixed TIMP-1 may represent a more effective version of the protein for use in therapeutic expression.
Subject(s)
Glycosylphosphatidylinositols/chemistry , Tissue Inhibitor of Metalloproteinase-1/metabolism , Tissue Inhibitor of Metalloproteinase-1/pharmacology , Animals , CHO Cells , Cell Line , Cell Membrane/drug effects , Cell Membrane/metabolism , Cell Movement/drug effects , Cell Movement/physiology , Cricetinae , Dose-Response Relationship, Drug , Endothelial Cells/drug effects , Endothelial Cells/physiology , Glycosylphosphatidylinositols/pharmacology , Humans , Matrix Metalloproteinase Inhibitors , Metalloendopeptidases/antagonists & inhibitors , Recombinant Fusion Proteins/metabolism , Recombinant Fusion Proteins/pharmacology , Surface PropertiesABSTRACT
Podocytes contribute to the filtration barrier within the kidney. The integrin-linked kinase (ILK) plays an important role in podocyte adhesion to the glomerular basement membrane, signal transduction and phenotype regulation. We demonstrate that ILK activity is also associated with upregulation of matrix metalloproteinase-9 (MMP-9) mRNA levels during podocyte stress. A synthetic ILK inhibitor blocked MMP-9 mRNA upregulation but showed no effect on TIMP-1 or MMP-2 mRNA expression. Interestingly, a corresponding increase in MMP-9 secretion was not observed, suggesting that MMP-9 mRNA production in podocytes is regulated via ILK, whereas additional signaling pathways may mediate the post-transcriptional regulation of MMP-9.