Identification of a peptide blocking vascular endothelial growth factor (VEGF)-mediated angiogenesis

Vascular endothelial growth factor (VEGF) binding to the kinase domain receptor (KDR/FLK1 or VEGFR-2) mediates vascularization and tumor-induced angiogenesis. Since there is evidence that KDR plays an important role in tumor angiogenesis, we sought to identify peptides able to block the VEGF-KDR interaction. A phage epitope library was screened by affinity for membrane-expressed KDR or for an anti-VEGF neutralizing monoclonal antibody. Both strategies led to the isolation of peptides binding KDR specifically, but those isolated by KDR binding tended to display lower reactivities. Of the synthetic peptides corresponding to selected clones tested to determine their inhibitory activity, ATWLPPR completely abolished VEGF binding to cell-displayed KDR. In vitro, this effect led to the inhibition of the VEGF-mediated proliferation of human vascular endothelial cells, in a dose-dependent and endothelial cell type-specific manner. Moreover, in vivo, ATWLPPR totally abolished VEGF-induced angiogenesis in a rabbit corneal model. Taken together, these data demonstrate that ATWLPPR is an effective antagonist of VEGF binding, and suggest that this peptide may be a potent inhibitor of tumor angiogenesis and metastasis.     

TTAC-0001, a human monoclonal antibody targeting VEGFR-2/KDR,blocks tumor angiogenesis

Angiogenesis is one of the most important processes for cancer cell survival, tumor growth and metastasis. Vascular endothelial growth factor (VEGF) and its receptor, particularly VEGF receptor-2 (VEGFR-2, or kinase insert domain-containing receptor, KDR), play critical roles in tumor-associated angiogenesis. We developed TTAC-0001, a human monoclonal antibody against VEGFR-2/KDR from a fully human naïve single-chain variable fragment phage library. TTAC-0001 was selected as a lead candidate based on its affinity, ligand binding inhibition and inhibition of VEGFR-2 signal in human umbilical vein endothelial cells (HUVEC). TTAC-0001 inhibited binding of VEGF-C and VEGF-D to VEGFR-2 in addition to VEGF-A. It binds on the N-terminal regions of domain 2 and domain 3 of VEGFR-2. It could inhibit the phosphorylation of VEGFR-2/KDR and ERK induced by VEGF in HUVEC. TTAC-0001 also inhibited VEGF-mediated endothelial cell proliferation, migration and tube formation in vitro, as well as ex vivo vessel sprouting from rat aortic rings and neovascularization in mouse matrigel model in vivo. Our data indicates that TTAC-0001 blocks the binding of VEGFs to VEGFR-2/KDR and inhibits VEGFR-induced signaling pathways and angiogenesis. Therefore, these data strongly support the further development of TTAC-0001 as an anti-cancer agent in the clinic.     

Structural and functional uncoupling of the enzymatic and angiogenic inhibitory activities of tissue inhibitor of metalloproteinase-2 (TIMP-2): loop 6 is a novel angiogenesis inhibitor

Tissue inhibitors of metalloproteinases (TIMPs) regulate tumor growth, progression, and angiogenesis in a variety of experimental cancer models and in human malignancies. Results from numerous studies have revealed important differences between TIMP family members in their ability to inhibit angiogenic processes in vitro and angiogenesis in vivo despite their universal ability to inhibit matrix metalloproteinase (MMP) activity. To address these differences, a series of structure-function studies were conducted to identify and to characterize the anti-angiogenic domains of TIMP-2, the endogenous MMP inhibitor that uniquely inhibits capillary endothelial cell (EC) proliferation as well as angiogenesis in vivo. We demonstrate that the COOH-terminal domain of TIMP-2 (T2C) inhibits the proliferation of capillary EC at molar concentrations comparable with those previously reported for intact TIMP-2, while the NH2-terminal domain (T2N), which inhibits MMP activity, has no significant anti-proliferative effect. Interestingly, although both T2N and T2C inhibited embryonic angiogenesis, only T2C resulted in the potent inhibition of angiogenesis driven by the exogenous addition of angiogenic mitogen, suggesting that MMP inhibition alone may not be sufficient to inhibit the aggressive neovascularization characteristic of aberrant angiogenesis. We further mapped the anti-proliferative activity of T2C to a 24-amino acid peptide corresponding to Loop 6 of TIMP-2 and show that Loop 6 is a potent inhibitor of both embryonic and mitogen-stimulated angiogenesis in vivo. These findings demonstrate that TIMP-2 possesses two distinct types of anti-angiogenic activities which can be uncoupled from each other, the first represented by its MMP-dependent inhibitory activity which can inhibit only embryonic neovascularization and the second represented by an MMP-independent activity which inhibits both normal angiogenesis and mitogen-driven angiogenesis in vivo. In addition, we report, for the first time, the discovery of Loop 6 as a novel and potent inhibitor of angiogenesis.     

Alzheimer's β-amyloid peptide blocks vascular endothelial growth factor mediated signaling via direct interaction with VEGFR-2

Beta-amyloid peptides (Aβ) are the major constituents of senile plaques and cerebrovascular deposits in the brains of Alzheimer's disease patients. We have shown previously that soluble forms of Aβ are anti-angiogenic both in vitro and in vivo . However, the mechanism of the anti-angiogenic activity of Aβ peptides is unclear. In this study, we examined the effects of Aβ1–42 on vascular endothelial growth factor receptor 2 (VEGFR-2) signaling, which plays a key role in angiogenesis. Aβ inhibited VEGF-induced migration of endothelial cells, as well as VEGF-induced permeability of an in vitro model of the blood brain barrier. Consistently, exogenous VEGF dose-dependently antagonized the anti-angiogenic activity of Aβ in a capillary network assay. Aβ1–42 also blocked VEGF-induced tyrosine phosphorylation of VEGFR-2 in two types of primary endothelial cells, suggesting an antagonistic action of Aβ toward VEGFR-2 signaling in cells. Moreover, Aβ was able to directly interact with the extracellular domain of VEGFR-2 and to compete with the binding of VEGF to its receptor in a cell-free assay. Co-immunoprecipitation experiments confirmed that Aβ can bind VEGFR-2 both in vitro and in vivo . Altogether, our data suggest that Aβ acts as an antagonist of VEGFR-2 and provide a mechanism explaining the anti-angiogenic activity of Aβ peptides.     

A novel function for tissue inhibitor of metalloproteinases-3 (TIMP3): inhibition of angiogenesis by blockage of VEGF binding to VEGF receptor-2

Tissue inhibitor of metalloproteinases-3 (TIMP3) is one of four members of a family of proteins that were originally classified according to their ability to inhibit matrix metalloproteinases (MMP). TIMP3, which encodes a potent angiogenesis inhibitor, is mutated in Sorsby fundus dystrophy, a macular degenerative disease with submacular choroidal neovascularization. In this study we demonstrate the ability of TIMP3 to inhibit vascular endothelial factor (VEGF)-mediated angiogenesis and identify the potential mechanism by which this occurs: TIMP3 blocks the binding of VEGF to VEGF receptor-2 and inhibits downstream signaling and angiogenesis. This property seems to be independent of its MMP-inhibitory activity, indicating a new function for this molecule.     

A peptide encoded by exon 6 of VEGF (EG3306) inhibits VEGF-induced angiogenesis in vitro and ischaemic retinal neovascularisation in vivo

VEGF is an important mediator of pathological angiogenesis in the eye and is a target for the development of novel anti-angiogenic molecules. In a previous study we identified 12-amino acid peptides derived from exon 6 of VEGF that inhibited VEGF binding to its receptors in HUVECs, endothelial cell functions, and in vitro angiogenesis. Screening of a series of truncated peptides corresponding to the inhibitory region of exon 6 identified a seven amino acid residue peptide, RKRKKSR, as the minimum exon 6-encoded sequence which retains the ability to inhibit VEGF receptor binding and angiogenesis in vitro. The effect of the seven-residue peptide was examined in a mouse model of ischaemic retinal neovascularisation. Administration of the peptide caused a 50% inhibition of retinal neovascularisation and was as effective in inhibiting ischaemic angiogenesis as soluble Flt-1 adenovirus. These results demonstrate that a seven amino acid VEGF exon 6-derived peptide is an effective inhibitor of ocular neovascularisation in vivo, and may have applications in the treatment of pathophysiological ocular neovascularisation in human disease.     

Engineered conformation-dependent VEGF peptide mimics are effective in inhibiting VEGF signaling pathways

Angiogenesis, or formation of new blood vessels, is crucial to cancer tumor growth. Tumor growth, progression, and metastasis are critically influenced by the production of the pro-angiogenic vascular endothelial growth factor (VEGF). Promising anti-angiogenic drugs are currently available; however, their susceptibilities to drug resistance and long term toxicity are serious impediments to their use, thus requiring the development of new therapeutic approaches for safe and effective angiogenic inhibitors. In this work, peptides were designed to mimic the VEGF-binding site to its receptor VEGFR-2. The VEGF conformational peptide mimic, VEGF-P3(CYC), included two artificial cysteine residues, which upon cyclization constrained the peptide in a loop native-like conformation to better mimic the anti-parallel structure of VEGF. The engineered cyclic VEGF mimic peptide demonstrated the highest affinity to VEGFR-2 by surface plasmon resonance assay. The VEGF peptide mimics were evaluated as inhibitors in several in vitro assays in which VEGF-dependent signaling pathways were observed. All VEGF mimics inhibited VEGFR-2 phosphorylation with VEGF-P3(CYC) showing the highest inhibitory effects when compared with unstructured peptides. Additionally, we show in several angiogenic in vitro assays that all the VEGF mimics inhibited endothelial cell proliferation, migration, and network formation with the conformational VEGF-P3 (CYC) being the best. The VEGF-P3(CYC) also caused a significant delay in tumor development in a transgenic model of VEGF(+/-)Neu2-5(+/-). These results indicate that the structure-based design is important for the development of this peptidomimetic and for its anti-angiogenic effects.     

Ganglioside GM3 inhibits VEGF/VEGFR-2-mediated angiogenesis: direct interaction of GM3 with VEGFR-2

Angiogenesis is associated with growth, invasion, and metastasis of human solid tumors. Aberrant activation of endothelial cells and induction of microvascular permeability by a vascular endothelial growth factor (VEGF) receptor-2 (VEGFR-2) signaling pathway is observed in pathological angiogenesis including tumor, wound healing, arthritis, psoriasis, diabetic retinopathy, and others. Here, we show that GM3 regulated the activity of various downstream signaling pathways and biological events through the inhibition of VEGF-stimulated VEGFR-2 activation in vascular endothelial cells in vitro. Furthermore, GM3 strongly blocked VEGF-induced neovascularization in vivo, in models including the chick chorioallantoic membrane and Matrigel plug assay. Interestingly, GM3 suppressed VEGF-induced VEGFR-2 activation by blocking its dimerization and also blocked the binding of VEGF to VEGFR-2 through a GM3-specific interaction with the extracellular domain of VEGFR-2, but not with VEGF. Primary tumor growth in mice was inhibited by subcutaneous injection of GM3. Immunohistochemical analyses showed GM3 inhibition of angiogenesis and tumor cell proliferation. GM3 also resulted in the suppression of VEGF-stimulated microvessel permeability in mouse skin capillaries. These results suggest that GM3 inhibits VEGFR-2-mediated changes in vascular endothelial cell function and angiogenesis, and might be of value in anti-angiogenic therapy.     

Upregulation of neuropilin-1 by basic fibroblast growth factor enhances vascular smooth muscle cell migration in response to VEGF

Neuropilin-1 (NRP-1) is a co-receptor for vascular endothelial growth factor (VEGF). During neovascularization, vascular smooth muscle cells (VSMCs) and pericytes modulate the function of endothelial cells. Factors that mediate NRP-1 in human VSMCs (hVSMCs) remain to be elucidated. We studied various angiogenic cytokines to identify factors that increase NRP-1 expression in hVSMCs. Treatment of hVSMCs with basic fibroblast growth factor (b-FGF) induced expressions of NRP-1 mRNA and protein whereas epidermal growth factor, insulin-like growth factor-1, and interleukin-1beta did not. b-FGF induced phosphorylation of Erk-1/2 and JNK. MEK1/2 and nuclear factor kappa B (NF-kappaB) inhibitors (U0126 and TLCK, respectively) blocked the ability of b-FGF to induce NRP-1 mRNA expression, but inhibition of JNK (SP600125) or PI3-kinase activity (wortmannin) did not. Further, the increase in NRP-1 expression by b-FGF enhanced hVSMCs migration in response to VEGF(165). This effect was dependent on the binding of VEGF(165) to VEGFR-2, as blocking antibodies to VEGFR-2, but not VEGFR-1, inhibited VEGF(165)-induced migration. In conclusion, b-FGF increased NRP-1 expression in hVSMCs that in turn enhance the effect of VEGF(165) on cell migration. The enhanced migration of hVSMCs was mediated through binding of VEGF(165) to both NRP-1 and VEGFR-2, as inhibition of VEGFR-2 on these cells blocked the effect of VEGF-mediated cell migration.     

The C-terminus of viral vascular endothelial growth factor-E partially blocks binding to VEGF receptor-1

Vascular endothelial growth factor (VEGF) family members play important roles in embryonic development and angiogenesis during wound healing and in pathological conditions such as tumor formation. Parapoxviruses express a new member of the VEGF family which is a functional mitogen that specifically activates VEGF receptor (VEGFR)-2 but not VEGFR-1. In this study, we show that deletion from the viral VEGF of a unique C-terminal region increases both VEGFR-1 binding and VEGFR-1-mediated monocyte migration. Enzymatic removal of O-linked glycosylation from the C-terminus also increased VEGFR-1 binding and migration of THP-1 monocytes indicating that both the C-terminal residues and O-linked sugars contribute to blocking viral VEGF binding to VEGFR-1. The data suggest that conservation of the C-terminal residues throughout the viral VEGF subfamily may represent a means of reducing the immunostimulatory activities associated with VEGFR-1 activation while maintaining the ability to induce angiogenesis via VEGFR-2.     

The anti-angiogenic peptide, loop 6, binds insulin-like growth factor-1 receptor

Tissue inhibitors of metalloproteinases (TIMPs), the endogenous inhibitors of matrix metalloproteinases, have been shown to possess biological functions that are independent of their ability to inhibit matrix metalloproteinases. We have previously shown that the C-terminal domain of TIMP-2 and, in particular, Loop 6 inhibit capillary endothelial cell proliferation and angiogenesis both in vitro and in vivo. To elucidate the mechanism by which Loop 6 inhibits angiogenesis, we sought to determine whether its biological effects were the result of a known TIMP-2 protein-protein interaction or of a receptor-mediated event. In this study, we identify insulin-like growth factor-1 receptor as a binding partner of Loop 6/TIMP-2 and characterize this interaction on the endothelial cell surface and the consequences of this interaction on downstream receptor signaling.     

BRN-103, a novel nicotinamide derivative, inhibits VEGF-induced angiogenesis and proliferation in human umbilical vein endothelial cells

Anti-angiogenesis is regarded as an effective strategy for cancer treatment, and vascular endothelial growth factor (VEGF) plays a key role in the regulations of angiogenesis and vasculogenesis. In the present study, the authors synthesized five novel nicotinamide derivatives which structurally mimic the receptor tyrosine kinase inhibitor sunitinib and evaluated their anti-angiogenic effects. Transwell migration assays revealed that 2-(1-benzylpiperidin-4-yl) amino-N-(3-chlorophenyl) nicotinamide (BRN-103), among the five derivatives most potently inhibited VEGF-induced human umbilical vein endothelial cells (HUVECs). In addition, BRN-103 dose-dependently inhibited VEGF-induced migration, proliferation, and capillary-like tube formation of HUVECs and vessel sprouting from mouse aortic rings. To understand the molecular mechanisms responsible for these activities, the authors examined the effect of BRN-103 on VEGF signaling pathways in HUVECs. BRN-103 was found to suppress the VEGF-induced phosphorylation of VEGF receptor 2 (VEGR2) and the activations of AKT and eNOS. Taken together, these results suggest that BRN-103 inhibits VEGF-mediated angiogenesis signaling in human endothelial cells.     

Structure and inhibitory effects on angiogenesis and tumor development of a new vascular endothelial growth inhibitor

Blocking angiogenesis is an attractive strategy to inhibit tumor growth, invasion, and metastasis. We describe here the structure and the biological action of a new cyclic peptide derived from vascular endothelial growth factor (VEGF). This 17-amino acid molecule designated cyclopeptidic vascular endothelial growth inhibitor (cyclo-VEGI, CBO-P11) encompasses residues 79-93 of VEGF which are involved in the interaction with VEGF receptor-2. In aqueous solution, cyclo-VEGI presents a propensity to adopt a helix conformation that was largely unexpected because only beta-sheet structures or random coil conformations have been observed for macrocyclic peptides. Cyclo-VEGI inhibits binding of iodinated VEGF165 to endothelial cells, endothelial cells proliferation, migration, and signaling induced by VEGF165. This peptide also exhibits anti-angiogenic activity in vivo on the differentiated chicken chorioallantoic membrane. Furthermore, cyclo-VEGI significantly blocks the growth of established intracranial glioma in nude and syngeneic mice and improves survival without side effects. Taken together, these results suggest that cyclo-VEGI is an attractive candidate for the development of novel angiogenesis inhibitor molecules useful for the treatment of cancer and other angiogenesis-related diseases.     

A mutant form of vascular endothelial growth factor (VEGF) that lacks VEGF receptor-2 activation retains the ability to induce vascular permeability

Vascular endothelial growth factor (VEGF) is a major mediator of vasculogenesis and angiogenesis both during development and in pathological conditions. VEGF has a variety of effects on vascular endothelium, including the ability to stimulate endothelial cell mitogenesis, and the potent induction of vascular permeability. These activities are at least in part mediated by binding to two high affinity receptors, VEGFR-1 and VEGFR-2. In this study we have made mutations of mouse VEGF in order to define the regions that are required for VEGFR-2-mediated functions. Development of a bioassay, which responds only to signals generated by cross-linking of VEGFR-2, has allowed evaluation of these mutants for their ability to activate VEGFR-2. One mutant (VEGF0), which had amino acids 83-89 of VEGF substituted with the analogous region of the related placenta growth factor, demonstrated significantly reduced VEGFR-2 binding compared with wild type VEGF, indicating that this region was required for VEGF-VEGFR-2 interaction. Intriguingly, when this mutant was evaluated in a Miles assay for its ability to induce vascular permeability, no difference was found when compared with wild type VEGF. In addition we have shown that the VEGF homology domain of the structurally related growth factor VEGF-D is capable of binding to and activating VEGFR-2 but has no vascular permeability activity, indicating that VEGFR-2 binding does not correlate with permeability activity for all VEGF family members. These data suggest different mechanisms for VEGF-mediated mitogenesis and vascular permeability and raise the possibility of an alternative receptor mediating vascular permeability.     

The Polyclonal Antibodies Induced by VBP3 Complex Peptide Targeting Angiogenesis and Tumor Suppression

Basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) play a critical role in tumor-associated angiogenesis and have become the targets of anti-tumor therapy. The BALB/c mice were immunized with VEGF/bFGF complex peptide (VBP3) constructed with different epitope peptides of human VEGF and bFGF. The results of the immunogenicity showed that the VBP3 could effectively stimulate immune response in mice and elicit the mice to produce high titer specific anti-VEGF and anti-bFGF antibodies (anti-VBP3 antibodies). The polyclonal anti-VBP3 antibodies separated from the mouse immune serum could effectively inhibit the proliferation, migration and tube formation of human umbilical vein endothelial cells (HUVECs) and block the proliferation and migration of lung cancer A549 cells. Besides, the anti-VBP3 antibodies could effectively inhibit tumor growth and tumor angiogenesis in BABL/c nude mice. The results demonstrated that the VBP3 complex peptide could elicit the body to produce the high titer anti-VEGF and anti-bFGF antibodies, which showed anti-tumor and anti-angiogenic effects in vitro and in vivo. The results revealed that the VBP3 complex peptide could be used as a potential peptide vaccine in tumor therapy.     

Identification of PDCL3 as a Novel Chaperone Protein Involved in the Generation of Functional VEGF Receptor 2

Angiogenesis, a hallmark step in tumor metastasis and ocular neovascularization, is driven primarily by the function of VEGF ligand on one of its receptors, VEGF receptor 2 (VEGFR-2). Central to the proliferation and ensuing angiogenesis of endothelial cells, the abundance of VEGFR-2 on the surface of endothelial cells is essential for VEGF to recognize and activate VEGFR-2. We have identified phosducin-like 3 (PDCL3, also known as PhLP2A), through a yeast two-hybrid system, as a novel protein involved in the stabilization of VEGFR-2 by serving as a chaperone. PDCL3 binds to the juxtamembrane domain of VEGFR-2 and controls the abundance of VEGFR-2 by inhibiting its ubiquitination and degradation. PDCL3 increases VEGF-induced tyrosine phosphorylation and is required for VEGFR-2-dependent endothelial capillary tube formation and proliferation. Taken together, our data provide strong evidence for the role of PDCL3 in angiogenesis and establishes the molecular mechanism by which it regulates VEGFR-2 expression and function.     

Vascular endothelial growth factor (VEGF)/VEGF-C mosaic molecules reveal specificity determinants and feature novel receptor binding patterns

Vascular endothelial growth factors (VEGFs) and their receptors play key roles in angiogenesis and lymphangiogenesis. VEGF activates VEGF receptor-1 (VEGFR-1) and VEGFR-2, whereas VEGF-C activates VEGFR-2 and VEGFR-3. We have created a library of VEGF/VEGF-C mosaic molecules that contains factors with novel receptor binding profiles, notably proteins binding to all three VEGF receptors ("super-VEGFs"). The analyzed super-VEGFs show both angiogenic and lymphangiogenic effects in vivo, although weaker than the parental molecules. The composition of the VEGFR-3 binding molecules and scanning mutagenesis revealed determinants of receptor binding and specificity. VEGFR-2 and VEGFR-3 showed striking differences in their requirements for VEGF-C binding; extracellular domain 2 of VEGFR-2 was sufficient, whereas in VEGFR-3, both domains 1 and 2 were necessary.     

TIMP-1 promotes VEGF-induced neovascularization in the retina

Proteolysis of vascular basement membranes and surrounding extracellular matrix is a critical early step in neovascularization. It requires alteration of the balance between matrix metalloproteinases (MMPs) and proteins that bind to and inactivate MMPs, tissue inhibitors of metalloproteinases (TIMPs). TIMP-1 has been demonstrated to inhibit neovascularization in chick chorioallantoic membranes. However, TIMP-1 has also been shown to either promote or inhibit cell proliferation and migration in different settings. To determine whether genetic alteration of the MMP/TIMP-1 ratio would alter retinal neovascularization, we crossed mice that express vascular endothelial growth factor (VEGF) in photoreceptors with TIMP-1-deficient mice or mice that overexpress TIMP-1. Compared to VEGF transgene-positive/TIMP-1-sufficient mice, VEGF transgene-positive/TIMP-1-deficient mice showed smaller neovascular lesions. There was also no difference between the two groups of mice in the appearance of the neovascularization by light or electron microscopy. Compound VEGF/TIMP-1 transgenic mice had increased expression of both VEGF and TIMP-1 in the retina, and had more neovascularization than mice that had increased expression of VEGF alone. These gain- and loss-of-function data suggest that alteration of the TIMP-1/MMP ratio modulates retinal neovascularization in a complex manner and not simply by altering the proteolytic activity and thereby invasiveness of endothelial cells.     

An integrin-binding N-terminal peptide region of TIMP-2 retains potent angio-inhibitory and anti-tumorigenic activity in vivo

Tissue inhibitor of metalloproteinases-2 (TIMP-2) inhibits angiogenesis by several mechanisms involving either MMP inhibition or direct endothelial cell binding. The primary aim of this study was to identify the TIMP-2 region involved in binding to the previously identified receptor integrin α3β1, and to determine whether synthetic peptides derived from this region retained angio-inhibitory and tumor suppressor activity. We demonstrated that the N-terminal domain of TIMP-2 (N-TIMP-2) binds to α3β1 and inhibits vascular endothelial growth factor-stimulated endothelial cell growth in vitro, suggesting that both the α3β1-binding domain and the growth suppressor activity of TIMP-2 localize to the N-terminal domain. Using a peptide array approach we identify a 24 amino acid region of TIMP-2 primary sequence, consisting of residues Ile43-Ala66, which shows α3β1-binding activity. Subsequently we demonstrate that synthetic peptides from this region compete for TIMP-2 binding to α3β1 and suppress endothelial growth in vitro. We define a minimal peptide sequence (peptide 8-9) that possesses both angio-inhibitory and, using a murine xenograft model of Kaposi's sarcoma, anti-tumorigenic activity in vivo. Thus, both the α3β1-binding and the angio-inhibitory activities co-localize to a solvent exposed, flexible region in the TIMP-2 primary sequence that is unique in amino acid sequence compared with other members of the TIMP family. Furthermore, comparison of the TIMP-2 and TIMP-1 protein 3-D structures in this region also identified unique structural differences. Our findings demonstrate that the integrin binding, tumor growth suppressor and in vivo angio-inhibitory activities of TIMP-2 are intimately associated within a unique sequence/structural loop (B-C loop).