Recombinant GPI-Anchored TIMP-1 Stimulates Growth and Migration of Peritoneal Mesothelial Cells

Abstract

BackgroundMesothelial cells are critical in the pathogenesis of post-surgical intraabdominal adhesions as well as in the deterioration of the peritoneal membrane associated with long-term peritoneal dialysis. Mesothelial denudation is a pathophysiolocigally important finding in these processes. Matrix metalloproteinase (MMP) biology underlies aspects of mesothelial homeostasis as well as wound repair. The endogenous tissue inhibitors of metalloproteinases (TIMPs) moderate MMP activity.Methods and FindingBy modifying human TIMP-1 through the addition of a glycosylphosphatidylinositol (GPI) anchor, a recombinant protein was generated that efficiently focuses TIMP-1 on the cell surface. Treatment of primary mesothelial cells with TIMP-1-GPI facilitates their mobilization and migration leading to a dramatic increase in the rate of wound experimental closure. Mesothelial cells treated with TIMP-1-GPI showed a dose dependent increase in cell proliferation, reduced secretion of MMP-2, MMP-9, TNF-α and urokinase-type plasminogen activator (uPA), but increased tissue plasminogen activator (t-PA). Treatment resulted in reduced expression and processing of latent TGF-β1.ConclusionsTIMP-1-GPI stimulated rapid and efficient in vitro wound closure. The agent enhanced mesothelial cell proliferation and migration and was bioactive in the nanogram range. The application of TIMP-1-GPI may represent a new approach for limiting or repairing damaged mesothelium.

Treatment of Dermal Fibroblasts with GPI-Anchored Human TIMP-1 Protein Moderates Processes Linked to Scar Formation

“…A reduction in MMP activity by increased levels of TIMP-1 at the cell surface should result in a more pronounced accumulation of matrix components because of a blockade in their catabolism. The effects seen may be linked to the observed reduced expression of tgf-b1 mRNA and protein resulting from treatment (Djafarzadeh et al, 2012). TGF-b1 is known to stimulate the proliferation of fibroblasts (Moses et al, 1990;Verrecchia et al, 2001;Leask and Abraham, 2004).…”

“…The bioactivity of TIMP-1 can be altered by changing its subcellular distribution (Djafarzadeh et al, 2004(Djafarzadeh et al, , 2006(Djafarzadeh et al, , 2012Raggi et al, 2009). Human TIMP-1 protein was modified by fusion to a glycosylphosphatidylinositol (GPI) anchor.…”

“…Recombinant TIMP-1-GPI is efficiently incorporated into cell membranes where it results in a shift in the proteolytic microenvironment at the cell surface. This can result in enhanced and new cellular behavior (Djafarzadeh et al, 2004(Djafarzadeh et al, , 2006(Djafarzadeh et al, , 2012Raggi et al, 2009).…”

Cell surface engineering of renal cell carcinoma with glycosylphosphatidylinositol-anchored TIMP-1 blocks TGF-β1 activation and reduces regulatory ID gene expression

“…TIMP-1 -GPI was originally developed to deliver TIMP-1 to defined tissue environments. By insertion into cell membranes, the GPI anchor limits diffusion of the fusion protein from the site of application (Djafarzadeh et al , 2004(Djafarzadeh et al , , 2006(Djafarzadeh et al , , 2012Raggi et al , 2009 ). Intraoperative peritumoral application of TIMP-1 -GPI as an adjuvant to surgery could help maintain tumor control by targeting microscopic residual tumor cells in the context of resection.…”

“…This modification effectively shifts the functional activity of TIMP-1 from the ECM directly on to the cell surface and alters proteolytic dynamics at the cell membrane (Figure 1 A). Previous work has shown that TIMP-1 -GPI treatment can result in enhanced biological effects (Djafarzadeh et al , 2004(Djafarzadeh et al , , 2006(Djafarzadeh et al , , 2012Raggi et al , 2009 ). These include changes in cellular proliferation (Djafarzadeh et al , 2004(Djafarzadeh et al , , 2006(Djafarzadeh et al , , 2012, and in the case of RCC an increased sensitivity to Fas-mediated apoptosis, reduced proliferation, migration and capacity for invasion (Djafarzadeh et al , 2006 ).…”