Abstract
Tissue inhibitors of metalloproteinases (TIMPs) while originally characterized as inhibitors of matrix metalloproteinases (MMPs) have recently been shown to have a wide range of functions that are independent of their MMP inhibitory properties. Tissue inhibitor of metalloproteinases-3 (TIMP-3) is a potent inhibitor of VEGF-mediated angiogenesis and neovascularization through its ability to block the binding of VEGF to its receptor VEGFR-2. To identify and characterize the anti-angiogenic domain of TIMP-3, structure function analyses and synthetic peptide studies were performed using VEGF-mediated receptor binding, signaling, migration and proliferation. In addition, the ability of TIMP-3 peptides to inhibit CNV in a mouse model was evaluated. We demonstrate that the anti-angiogenic property resides in the COOH-terminal domain of TIMP-3 protein which can block the binding of VEGF specifically to its receptor VEGFR-2, but not to VEGFR-1 similar to the full-length wild-type protein. Synthetic peptides corresponding to putative loop 6 and tail region of TIMP-3 have anti-angiogenic properties as determined by inhibition of VEGF binding to VEGFR-2, VEGF-induced phosphorylation of VEGFR-2 and downstream signaling pathways as well as endothelial cell proliferation and migration in response to VEGF. In addition, we show that intravitreal administration of TIMP-3 peptide could inhibit the size of laser-induced choroidal neovascularization lesions in mice. Thus, we have identified TIMP-3 peptides to be efficient inhibitors of angiogenesis and have a potential to be used therapeutically in diseases with increased neovascularization.
Highlights
Tissue inhibitors of metalloproteinases (TIMPs) constitute a family of four proteins (TIMP-1, TIMP-2, Tissue inhibitor of metalloproteinases-3 (TIMP-3) and TIMP-4) that are endogenous inhibitors of matrix (MMP) and play a critical role in the maintenance of extracellular matrix (ECM) homeostasis
We have previously demonstrated that TIMP-3 is a potent angiogenesis inhibitor, and functions independently of its matrix metalloproteinases (MMPs) inhibitory activity in this regard, by blocking the binding of vascular endothelial growth factor (VEGF) to its receptor VEGFR-2 [8]
COOH-terminal TIMP-3 Domain Inhibits VEGF Binding to VEGFR-2
Summary
Tissue inhibitors of metalloproteinases (TIMPs) constitute a family of four proteins (TIMP-1, TIMP-2, TIMP-3 and TIMP-4) that are endogenous inhibitors of matrix (MMP) and play a critical role in the maintenance of extracellular matrix (ECM) homeostasis. Originally characterized for their functional property to inhibit MMP activity, TIMPs have more recently been shown to have additional biological activities that may be independent of their MMP-inhibitory functions [7]. We have previously demonstrated that TIMP-3 is a potent angiogenesis inhibitor, and functions independently of its MMP inhibitory activity in this regard, by blocking the binding of vascular endothelial growth factor (VEGF) to its receptor VEGFR-2 [8]. To identify the anti-angiogenic functional domains of TIMP-3, we performed a series of structure-function analyses examining VEGF binding to VEGFR-2, and downstream endothelial cell proliferation and migration. We determined that the COOH-terminal domain of TIMP-3 contains the angioinhibitory activity with the NH2-terminal domain being inert for this function. We further mapped the ability to block VEGF binding to VEGFR-2 to the ‘‘loop 6’’ and ‘‘tail’’ peptides and the capability of ‘‘loop 6’’ to inhibit choroidal neovascularization (CNV) in a rodent model
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