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Tissue inhibitor of metalloproteinases-3 peptides inhibit angiogenesis and choroidal neovascularization in mice.

Qi JH, Ebrahem Q, Ali M, Cutler A, Bell B, Prayson N, Sears J, Knauper V, Murphy G, Anand-Apte B - PLoS ONE (2013)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, Cole Eye Institute, Cleveland Clinic Lerner College of Medicine at Case Western Reserve University, Cleveland, Ohio, United States of America.

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.

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The effect of synthetic TIMP-3 peptides on binding of 50 ng/ml soluble (s) VEGFR-1 or –2 to immobilized VEGF.A competitive ELISA assay was used as described under “Materials and Methods” (a). Loop 6 and tail peptides inhibit sVEGFR-2-VEGF complexes in a concentration-dependent manner. (b) Loop5 and N-peptide have no effect on sVEGFR-2-VEGF complexes. (c) Loop 5, loop 6, tail and N-peptide are unable to inhibit sVEGFR-1-VEGF complexes.
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pone-0055667-g002: The effect of synthetic TIMP-3 peptides on binding of 50 ng/ml soluble (s) VEGFR-1 or –2 to immobilized VEGF.A competitive ELISA assay was used as described under “Materials and Methods” (a). Loop 6 and tail peptides inhibit sVEGFR-2-VEGF complexes in a concentration-dependent manner. (b) Loop5 and N-peptide have no effect on sVEGFR-2-VEGF complexes. (c) Loop 5, loop 6, tail and N-peptide are unable to inhibit sVEGFR-1-VEGF complexes.

Mentions: VEGF-A signals primarily via the VEGF-receptor tyrosine kinases, VEGFR-1 and 2, with VEGFR-2 being implicated as the predominant pro-angiogenic receptor. The role of VEGFR-1 has been more controversial with some data demonstrating it as having a pro-angiogenic role [16] and others an anti-angiogenic function [17]. The T3-peptides were initially tested for their ability to block the binding of VEGF to sVEGFR-2/Fc chimera in an in vitro competitive ELISA. T3-loop 6 and T3-tail could inhibit binding of VEGF to sVEGFR-2/Fc chimera (Fig. 2a) in a concentration dependent fashion with an IC50 of 2 and 5 µM respectively. In contrast, two peptides corresponding to T3-loop 5 and the NH2-terminal domain of intact TIMP-3 (N-peptide) did not show any inhibitory activity (Fig. 2b). Interestingly, the IC 50 of the small peptides was in the µM range compared with the nM range doses seen with the entire COOH-terminal T3 domain. In addition, as observed with the COOH-terminal T3 domain, T3-loop 6 and T3-tail did not inhibit VEGF binding to VEGFR-1 (Flt-1) (Fig. 2c). Whether pan-inhibition of VEGF is necessary for inhibition of neovascularization or if inhibition of signaling via VEGFR-2 is sufficient has not been ascertained. Based on our results with TIMP-3 peptides we postulated that the TIMP-3 would be a useful tool to determine if specific inhibition of binding of VEGF to VEGFR-2 could inhibit VEGF-mediated angiogenesis.


Tissue inhibitor of metalloproteinases-3 peptides inhibit angiogenesis and choroidal neovascularization in mice.

Qi JH, Ebrahem Q, Ali M, Cutler A, Bell B, Prayson N, Sears J, Knauper V, Murphy G, Anand-Apte B - PLoS ONE (2013)

The effect of synthetic TIMP-3 peptides on binding of 50 ng/ml soluble (s) VEGFR-1 or –2 to immobilized VEGF.A competitive ELISA assay was used as described under “Materials and Methods” (a). Loop 6 and tail peptides inhibit sVEGFR-2-VEGF complexes in a concentration-dependent manner. (b) Loop5 and N-peptide have no effect on sVEGFR-2-VEGF complexes. (c) Loop 5, loop 6, tail and N-peptide are unable to inhibit sVEGFR-1-VEGF complexes.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3585964&req=5

pone-0055667-g002: The effect of synthetic TIMP-3 peptides on binding of 50 ng/ml soluble (s) VEGFR-1 or –2 to immobilized VEGF.A competitive ELISA assay was used as described under “Materials and Methods” (a). Loop 6 and tail peptides inhibit sVEGFR-2-VEGF complexes in a concentration-dependent manner. (b) Loop5 and N-peptide have no effect on sVEGFR-2-VEGF complexes. (c) Loop 5, loop 6, tail and N-peptide are unable to inhibit sVEGFR-1-VEGF complexes.
Mentions: VEGF-A signals primarily via the VEGF-receptor tyrosine kinases, VEGFR-1 and 2, with VEGFR-2 being implicated as the predominant pro-angiogenic receptor. The role of VEGFR-1 has been more controversial with some data demonstrating it as having a pro-angiogenic role [16] and others an anti-angiogenic function [17]. The T3-peptides were initially tested for their ability to block the binding of VEGF to sVEGFR-2/Fc chimera in an in vitro competitive ELISA. T3-loop 6 and T3-tail could inhibit binding of VEGF to sVEGFR-2/Fc chimera (Fig. 2a) in a concentration dependent fashion with an IC50 of 2 and 5 µM respectively. In contrast, two peptides corresponding to T3-loop 5 and the NH2-terminal domain of intact TIMP-3 (N-peptide) did not show any inhibitory activity (Fig. 2b). Interestingly, the IC 50 of the small peptides was in the µM range compared with the nM range doses seen with the entire COOH-terminal T3 domain. In addition, as observed with the COOH-terminal T3 domain, T3-loop 6 and T3-tail did not inhibit VEGF binding to VEGFR-1 (Flt-1) (Fig. 2c). Whether pan-inhibition of VEGF is necessary for inhibition of neovascularization or if inhibition of signaling via VEGFR-2 is sufficient has not been ascertained. Based on our results with TIMP-3 peptides we postulated that the TIMP-3 would be a useful tool to determine if specific inhibition of binding of VEGF to VEGFR-2 could inhibit VEGF-mediated angiogenesis.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, Cole Eye Institute, Cleveland Clinic Lerner College of Medicine at Case Western Reserve University, Cleveland, Ohio, United States of America.

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.

Show MeSH
Related in: MedlinePlus