RESUMO
There is increasing appreciation for the role of the neurovascular unit in neurodegenerative diseases. We showed previously that the angiogenic and neurotrophic cytokine, vascular endothelial growth factor (VEGF), is suppressed to abnormally low levels in spinocerebellar ataxia type 1 (SCA1), and that replenishing VEGF reverses the cerebellar pathology in SCA1 mice. In that study, however, we used a recombinant VEGF, which is extremely costly to manufacture and biologically unstable as well as immunogenic. To develop a more viable therapy, here we test a synthetic VEGF peptide amphiphile that self-assembles into nanoparticles. We show that this nano-VEGF has potent neurotrophic and angiogenic properties, is well-tolerated, and leads to functional improvement in SCA1 mice even when administered at advanced stages of the disease. This approach can be generalized to other neurotrophic factors or molecules that act in a paracrine manner, offering a novel therapeutic strategy for neurodegenerative conditions.
Assuntos
Nanopartículas/administração & dosagem , Ataxias Espinocerebelares/tratamento farmacológico , Fator A de Crescimento do Endotélio Vascular/administração & dosagem , Adulto , Animais , Feminino , Técnicas de Introdução de Genes , Humanos , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Nanopartículas/química , Técnicas de Cultura de Órgãos , Ataxias Espinocerebelares/genética , Ataxias Espinocerebelares/fisiopatologia , Fator A de Crescimento do Endotélio Vascular/síntese químicaRESUMO
Total chemical synthesis was used to prepare the mirror image (D-protein) form of the angiogenic protein vascular endothelial growth factor (VEGF-A). Phage display against D-VEGF-A was used to screen designed libraries based on a unique small protein scaffold in order to identify a high affinity ligand. Chemically synthesized D- and L- forms of the protein ligand showed reciprocal chiral specificity in surface plasmon resonance binding experiments: The L-protein ligand bound only to D-VEGF-A, whereas the D-protein ligand bound only to L-VEGF-A. The D-protein ligand, but not the L-protein ligand, inhibited the binding of natural VEGF(165) to the VEGFR1 receptor. Racemic protein crystallography was used to determine the high resolution X-ray structure of the heterochiral complex consisting of {D-protein antagonist + L-protein form of VEGF-A}. Crystallization of a racemic mixture of these synthetic proteins in appropriate stoichiometry gave a racemic protein complex of more than 73 kDa containing six synthetic protein molecules. The structure of the complex was determined to a resolution of 1.6 Å. Detailed analysis of the interaction between the D-protein antagonist and the VEGF-A protein molecule showed that the binding interface comprised a contact surface area of approximately 800 Å(2) in accord with our design objectives, and that the D-protein antagonist binds to the same region of VEGF-A that interacts with VEGFR1-domain 2.
Assuntos
Modelos Moleculares , Complexos Multiproteicos/química , Fator A de Crescimento do Endotélio Vascular/química , Fator A de Crescimento do Endotélio Vascular/síntese química , Fator A de Crescimento do Endotélio Vascular/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Cromatografia Líquida de Alta Pressão , Dicroísmo Circular , Cristalografia por Raios X , Ensaio de Imunoadsorção Enzimática , Mapeamento de Epitopos , Ligantes , Espectrometria de Massas , Dados de Sequência Molecular , Complexos Multiproteicos/metabolismo , Fases de Leitura Aberta , Estereoisomerismo , Ressonância de Plasmônio de Superfície , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
Efficient access: the 204-residue covalent-dimer vascular endothelial growth factor with full mitogenic activity was prepared from three unprotected peptide segments by one-pot native chemical ligations. The covalent structure of the synthetic VEGF was confirmed by precise mass measurement, and the three-dimensional structure of the synthetic protein was determined by high-resolution X-ray crystallography.
Assuntos
Fator A de Crescimento do Endotélio Vascular/síntese química , Sequência de Aminoácidos , Cromatografia Líquida de Alta Pressão , Cristalografia por Raios X , Dimerização , Espectrometria de Massas , Dados de Sequência Molecular , Peptídeos/síntese química , Peptídeos/química , Estrutura Terciária de Proteína , Fator A de Crescimento do Endotélio Vascular/químicaRESUMO
OBJECTIVE: To assess the role of active immunotherapy targeting VEGF with a peptide vaccine as a potential treatment for ovarian cancer. METHODS: A peptide vaccine targeting antigenic B-cell epitopes of VEGF were identified and linked to a promiscuous T-cell epitope. Elicited antibodies were assessed for their ability to recognize the VEGF protein, inhibit angiogenesis, inhibit the interaction of VEGF with its receptor, and inhibit cancer growth in mice. RESULTS: Following immunization, high-titered elicited antibodies were shown to be specific for the full-length VEGF protein by ELISA and Western blot. Anti-VEGF peptide antibodies inhibited cellular migration, proliferation, invasion, tube formation, and growth of aortic ring cultures. These antibodies inhibited the interaction between VEGF and its receptor (VEGFR2) in a concentration-dependent manner. Confirmation of this mechanism was demonstrated through inhibition of VEGFR2 phosphorylation following culture of human endothelial vein endothelial cells with anti-VEGF peptide antibodies. These antibodies were shown to inhibit ovarian cancer xenograft growth in a nude mouse model following intraperitoneal passive immunization. Active immunization with the VEGF peptide vaccine inhibited VEGF-dependent pancreatic islet cell tumor growth in RIP1-Tag2 transgenic mice and was associated with decreased vasculogenesis in these tumors compared with animals vaccinated with an irrelevant peptide. Active immunization also inhibited growth of tumors from a VEGF overexpressing ovarian cancer cell line, resulting in decreased tumor size and tumor vessel density compared with control mice. CONCLUSIONS: Active immunization with VEGF peptides elicits antibodies that inhibit tumor growth by blocking VEGF-dependent angiogenesis.
Assuntos
Vacinas Anticâncer/imunologia , Neoplasias Ovarianas/terapia , Fragmentos de Peptídeos/imunologia , Vacinas de Subunidades Antigênicas/imunologia , Fator A de Crescimento do Endotélio Vascular/imunologia , Sequência de Aminoácidos , Animais , Anticorpos/imunologia , Vacinas Anticâncer/administração & dosagem , Epitopos de Linfócito B/imunologia , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Dados de Sequência Molecular , Vírus da Pneumonia Murina , Neovascularização Patológica/imunologia , Neoplasias Ovarianas/irrigação sanguínea , Neoplasias Ovarianas/imunologia , Fragmentos de Peptídeos/síntese química , Coelhos , Vacinas de Subunidades Antigênicas/administração & dosagem , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/síntese química , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
INTRODUCTION: VEGF receptors play a key role in angiogenesis and are important targets for several approved and many experimental drugs. Imaging of VEGF receptor expression in malignant tumors would provide important information, which can influence patient management. The aim of this study was the development of an easy-to-label positron-emitting tracer for imaging VEGF receptors. The tracer is based on engineered single-chain VEGF (scVEGF), expressed with cysteine-containing fusion tag (Cys-tag) for site-specific conjugation of PEGylated bifunctional chelating agents, HBED-CC or NOTA, suitable for labeling with (68)Ga at ambient temperature. METHODS: scVEGF-PEG-HBED-CC was synthesized by activating a single carboxyl group of the [Fe(HBED-CC)](-) complex with N-hydroxysuccinimide. Reaction of the activated complex with NH(2)-PEG-maleimide was followed by site-specific conjugation of PEGylated chelator to a thiol group in Cys-tag of scVEGF. The scVEGF-PEG-NOTA conjugate was synthesized using NHS-PEG-maleimide and p-NH(2)-Bn-NOTA. (68)Ga complexation was performed in HEPES buffer (pH 4.2) at room temperature. The functional activity after labeling was tested by radioligand cell binding assays. Biodistribution and PET studies in tumor-bearing mice were performed after 1, 2, 3 and 4 h postinjection. RESULTS: The radiolabeling of scVEGF-PEG-HBED-CC proved more efficient than scVEGF-PEG-NOTA allowing to stop the reaction after 4 min (>97% radiochemical yield). Radioligand cell binding assays performed on HEK-293 cells overexpressing VEGFR-2 revealed no change in the binding properties of (68)Ga-radiolabeled scVEGF relative to other scVEGF-based tracers. Both tracers showed comparable results in biodistribution, such as tumor accumulation and low liver uptake. The tracers were stable in 50% human serum for at least 72 h. CONCLUSIONS: The conjugates scVEGF-PEG-HBED-CC and scVEGF-PEG-NOTA revealed comparable in vivo characteristics and allowed easy-to-perform labeling with high stability for fast [(68)Ga]PET imaging of VEGF receptors in angiogenic vasculature.