Characterization of the Interaction Between Endostatin Short Peptide and VEGF Receptor 3

Abstract

Corneal angiogenesis and lymphangiogenesis are induced by vascular endothelial growth factors (VEGFs) signaling through its receptors VEGFR-1, -2, and -3. Endostatin is a peptide antagonist of these receptors that causes inhibition of bFGF-induced corneal angiogenesis and lymphangiogenesis. Here we show that binding of VEGF-C and endostatin to recombinant VEGFR-3 is competitive. Alignments of the primary amino acid sequences of VEGF-C and the C-terminal endostatin peptide (mEP: LEQKAASCHNSYIVLCIENSFMTSFSK) identified two conserved cysteine residues separated by seven amino acids. Peptides of VEGF-C and mEP containing these conserved residues bound toVEGFR-3. However, substitution of alanine for either of the cysteines in the mEP peptide perturbed the secondary structure, and this mutated peptide was unable to bind to VEGFR-3. Analysis by surface plasmon resonance demonstrated that the binding of the mEP peptide for recombinant VEGFR-3 had a Ka of 1.41 × 107M−1s−1, Kd of 0.6718 s−1, and a KD of 4.78 10−8M. Characterization of the mechanism of endostatin binding to VEGFR-3 may lead to the development of novel therapies for lymphangiogenesis-related disorders, such as transplant rejection, lymphedema, and cancer metastasis.

Endostatin's emerging roles in angiogenesis, lymphangiogenesis, disease, and clinical applications

“…VEGFR-3 (also known as flt-4), another receptor tyrosine kinase, is expressed primarily on lymphatic endothelial cell surfaces [103]. Endostatin competitively inhibits VEGF binding to VEGFR-3 in vitro [104]. Once bound, endostatin serves as an anti-lymphangiogenic factor by inhibiting VEGF-stimulated lymphatic endothelial cell proliferation and migration [104].…”

“…Endostatin competitively inhibits VEGF binding to VEGFR-3 in vitro [104]. Once bound, endostatin serves as an anti-lymphangiogenic factor by inhibiting VEGF-stimulated lymphatic endothelial cell proliferation and migration [104]. …”

“…Our research group has employed SPR assays to evaluate the activity of various short endostatin peptides and further characterize how endogenous endostatin binds to its receptor [104]. We evaluated four short endostatin peptides (mEP, mEP-CA, mEP-AC, and mEP-AA), each 27 amino acids in length, and their specific affinity for the VEGFR-3 receptor in vitro [104].…”

CXCL10 suppression of hem- and lymph-angiogenesis in inflamed corneas through MMP13

“…It is also one of few tissues in the body that is normally devoid of any vascular blood or lymphatic structures. Studies have identified multiple mechanisms, including the expression of anti-angiogenic factors, lack of pro-angiogenic factors, and expression of soluble VEGF receptors as underlying mechanisms for maintaining cornea avascularity [1–5]. However, many pathological conditions such as trauma, chemical injury, inflammation, and infection can break the balance of pro- and anti- angiogenic actions, resulting in hem-angiogenesis (HG; the development of new blood vessels) and lymph-angiogenesis (LG; the development of new lymphatic vessels) [6–9].…”

Nanotechnology in Corneal Neovascularization Therapy—A Review

“…37,38 It is important to keep in mind that the counterbalancing mechanism that regulates angiogenesis is the overproduction of angiogenic stimulators (VEGF) and the underproduction of angiogenic inhibitors (e.g., angiostatin, endostatin). 15,[39][40][41] Because this balance is so delicate, therapeutic alternatives targeting angiogenic inhibitors are as essential as those targeting angiogenic stimulators. In one study of angiogenic inhibitors, Zhang et al examined the effects of plasminogen kringle 5 (K5), a proteolytic fragment of plasminogen that functions as a potent angiogenic inhibitor by preventing the proliferation of endothelial cells.…”