RESUMO
The Notch signaling pathway is an important therapeutic target for the treatment of inflammatory diseases and cancer. We previously created ligand-specific inhibitors of Notch signaling comprised of Fc fusions to specific EGF-like repeats of the Notch1 extracellular domain, called Notch decoys, which bound ligands, blocked Notch signaling, and showed anti-tumor activity with low toxicity. However, the study of their function depended on virally mediated expression, which precluded dosage control and limited clinical applicability. We have refined the decoy design to create peptibody-based Notch inhibitors comprising the core binding domains, EGF-like repeats 10-14, of either Notch1 or Notch4. These Notch peptibodies showed high secretion properties and production yields that were improved by nearly 100-fold compared to previous Notch decoys. Using surface plasmon resonance spectroscopy coupled with co-immunoprecipitation assays, we observed that Notch1 and Notch4 peptibodies demonstrate strong but distinct binding properties to Notch ligands DLL4 and JAG1. Both Notch1 and Notch4 peptibodies interfere with Notch signaling in endothelial cells and reduce expression of canonical Notch targets after treatment. While prior DLL4 inhibitors cause hyper-sprouting, the Notch1 peptibody reduced angiogenesis in a 3-dimensional in vitro sprouting assay. Administration of Notch1 peptibodies to neonate mice resulted in reduced radial outgrowth of retinal vasculature, confirming anti-angiogenic properties. We conclude that purified Notch peptibodies comprising EGF-like repeats 10-14 bind to both DLL4 and JAG1 ligands and exhibit anti-angiogenic properties. Based on their secretion profile, unique Notch inhibitory activities, and anti-angiogenic properties, Notch peptibodies present new opportunities for therapeutic Notch inhibition.
Assuntos
Inibidores da Angiogênese , Células Endoteliais , Receptor Notch1 , Receptor Notch4 , Animais , Camundongos , Inibidores da Angiogênese/genética , Inibidores da Angiogênese/metabolismo , Inibidores da Angiogênese/farmacologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Fator de Crescimento Epidérmico/metabolismo , Imunoprecipitação , Inflamação/tratamento farmacológico , Inflamação/genética , Inflamação/metabolismo , Ligantes , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/metabolismo , Receptor Notch1/antagonistas & inibidores , Receptor Notch1/genética , Receptor Notch1/metabolismo , Receptor Notch4/genética , Receptor Notch4/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Vasos Retinianos/efeitos dos fármacos , Ressonância de Plasmônio de SuperfícieRESUMO
Accumulated evidence has shown that pre-eclampsia (PE) is related to both maternal and utero-placental antiangiogenesis and inflammation. Remarkably, an elevated cell-free fetal DNA (cffDNA) level has been found in maternal circulation; however, it remains unclear whether this DNA can induce activation of cytosolic DNA sensor signaling pathways and lead to the development of PE. In this study, we found that trophoblast cells constitutively expressed the cytosolic DNA sensors, absent in melanoma 2 (AIM2) and interferon-inducible protein 16 (IFI16). The cffDNA and pro-inflammatory and antiangiogenic factors were present at higher concentrations in PE compared with the control group and correlated with the severity of PE. DNA stimulation significantly increased the AIM2 and IFI16 levels, consistent with the elevated AIM2 and IFI16 expression in women with PE, and elicited increased production of AIM2-mediated interleukin IL-8 (IL-8), IL-6 and CC chemokine ligand 2 (CCL2) and IFI16-mediated sEndoglin, sFlt-1 and CXCL10. Furthermore, enhancement of the inflammatory response was found to be induced by DNA exposure, but DNA exposure did not induce PE-like symptoms in pregnant mice. It is possible that elevated cffDNA could reflect the degree of placental damage and trigger cytosolic DNA sensor activation, which disrupts the immunity balance and, consequently, contributes to inflammatory and antiangiogenic responses. In conclusion, the results of this study suggest that circulating cffDNA levels are increased in preeclamptic women and act through AIM2 and IFI16 activation to promote the production of pro-inflammatory and antiangiogenic factors, which correlate with the severity of the disease, and may offer insights into the etiology and pathogenesis of PE.
Assuntos
Ácidos Nucleicos Livres/genética , Proteínas de Ligação a DNA/genética , Proteínas Nucleares/genética , Fosfoproteínas/genética , Placenta/metabolismo , Pré-Eclâmpsia/genética , Adulto , Inibidores da Angiogênese/genética , Ácidos Nucleicos Livres/sangue , Proteínas de Ligação a DNA/sangue , Feminino , Feto , Regulação da Expressão Gênica/genética , Humanos , Inflamação/sangue , Inflamação/genética , Inflamação/patologia , Proteínas Nucleares/sangue , Fosfoproteínas/sangue , Placenta/patologia , Pré-Eclâmpsia/sangue , Pré-Eclâmpsia/patologia , Gravidez , Transdução de Sinais/genética , Trofoblastos/metabolismo , Trofoblastos/patologia , Adulto JovemRESUMO
BACKGROUND: The anti-angiogenic fusion protein RBDV-IgG1 Fc (RBDV), which comprises the receptor-binding domain of vascular endothelial growth factor-A (VEGF-A), has shown antitumour effects by reducing angiogenesis in vivo. This study used the cationic lipoplex lipo-PEG-PEI-complex (LPPC) to simultaneously encapsulate both the RBDV targeting protein and the RBDV plasmid (pRBDV) without covalent bonds to assess VEGFR targeting gene therapy in mice with melanoma in vivo. RESULTS: LPPC protected the therapeutic transgene from degradation by DNase, and the LPPC/RBDV complexes could specifically target VEGFR-positive B16-F10 cells both in vitro and in vivo. With or without RBDV protein-targeting direction, the pRBDV-expressing RBDV proteins were expressed and reached a maximal concentration on the 7th day in the sera after transfection in vivo and significantly elicited growth suppression against B16-F10 melanoma but not IgG1 control proteins. In particular, LPPC/pRBDV/RBDV treatment with the targeting molecules dramatically inhibited B16-F10 tumour growth in vivo to provide better therapeutic efficacy than the treatments with gene therapy with IgG1 protein targeting or administration of a protein drug with RBDV. CONCLUSIONS: The simultaneous combination of the LPPC complex with pRBDV gene therapy and RBDV protein targeting might be a potential tool to conveniently administer targeted gene therapy for cancer therapy.
Assuntos
Inibidores da Angiogênese/genética , Terapia Genética/métodos , Lipossomos/química , Melanoma Experimental/terapia , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Células 3T3 , Animais , Linhagem Celular Tumoral , Proliferação de Células , Fragmentos Fc das Imunoglobulinas/genética , Fragmentos Fc das Imunoglobulinas/metabolismo , Masculino , Melanoma Experimental/mortalidade , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos C57BL , Plasmídeos/química , Plasmídeos/genética , Plasmídeos/uso terapêutico , Domínios Proteicos/genética , Receptores de Fatores de Crescimento do Endotélio Vascular/genética , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/isolamento & purificação , Taxa de Sobrevida , Transplante Homólogo , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Formation of the vasculature by angiogenesis is critical for proper development, but angiogenesis also contributes to the pathogenesis of various disorders, including cancer and cardiovascular diseases. Vascular endothelial zinc finger 1 (Vezf1), is a Krüppel-like zinc finger protein that plays a vital role in vascular development. However, the mechanism by which Vezf1 regulates this process is not fully understood. Here, we show that Vezf1-/- mouse embryonic stem cells (ESC) have significantly increased expression of a stem cell factor, Cbp/p300-interacting transactivator 2 (Cited2). Compared with WT ESCs, Vezf1-/- ESCs inefficiently differentiated into endothelial cells (ECs), which exhibited defects in the tube-formation assay. These defects were due to reduced activation of EC-specific genes concomitant with lower enrichment of histone 3 acetylation at Lys27 (H3K27) at their promoters. We hypothesized that overexpression of Cited2 in Vezf1-/- cells sequesters P300/CBP away from the promoters of proangiogenic genes and thereby contributes to defective angiogenesis in these cells. This idea was supported by the observation that shRNA-mediated depletion of Cited2 significantly reduces the angiogenic defects in the Vezf1-/- ECs. In contrast to previous studies that have focused on the role of Vezf1 as a transcriptional activator of proangiogenic genes, our findings have revealed a role for Vezf1 in modulating the expression of the antiangiogenic factor Cited2. Vezf1 previously has been characterized as an insulator protein, and our results now provide insights into the mechanism, indicating that Vezf1 can block inappropriate, nonspecific interactions of promoters with cis-located enhancers, preventing aberrant promoter activation.
Assuntos
Inibidores da Angiogênese/metabolismo , Diferenciação Celular , Células-Tronco Embrionárias/metabolismo , Endotélio Vascular/metabolismo , Regulação da Expressão Gênica , Fatores de Transcrição Kruppel-Like/fisiologia , Proteínas Repressoras/fisiologia , Transativadores/fisiologia , Inibidores da Angiogênese/genética , Animais , Células Cultivadas , Proteínas de Ligação a DNA , Células-Tronco Embrionárias/citologia , Endotélio Vascular/citologia , Camundongos , Camundongos Knockout , Regiões Promotoras Genéticas , Fatores de TranscriçãoRESUMO
Designed ankyrin repeat proteins (DARPins) can recognize targets with specificities and affinities that equal or surpass those of antibodies, but because of their robustness and extreme stability, they allow a multitude of more advanced formats and applications. This review highlights recent advances in DARPin design, illustrates their properties, and gives some examples of their use. In research, they have been established as intracellular, real-time sensors of protein conformations and as crystallization chaperones. For future therapies, DARPins have been developed by advanced, structure-based protein engineering to selectively induce apoptosis in tumors by uncoupling surface receptors from their signaling cascades. They have also been used successfully for retargeting viruses. In ongoing clinical trials, DARPins have shown good safety and efficacy in macular degeneration diseases. These developments all ultimately exploit the high stability, solubility, and aggregation resistance of these molecules, permitting a wide range of conjugates and fusions to be produced and purified.
Assuntos
Inibidores da Angiogênese/uso terapêutico , Repetição de Anquirina , Portadores de Fármacos , Desenho de Fármacos , Terapia Genética/métodos , Engenharia de Proteínas/métodos , Proteínas Recombinantes/uso terapêutico , Inibidores da Angiogênese/química , Inibidores da Angiogênese/genética , Inibidores da Angiogênese/metabolismo , Animais , Humanos , Modelos Moleculares , Biblioteca de Peptídeos , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Relação Estrutura-AtividadeRESUMO
Ocular neovascularization is a common pathological feature in diabetic retinopathy and neovascular age-related macular degeneration that can lead to severe vision loss. We evaluated the therapeutic efficacy of a novel endogenous inhibitor of angiogenesis, the calreticulin anti-angiogenic domain (CAD180), and its functional 112-residue fragment, CAD-like peptide 112 (CAD112), delivered using a self-complementary adeno-associated virus serotype 2 (scAAV2) in rodent models of oxygen-induced retinopathy and laser-induced choroidal neovascularization. The expression of CAD180 and CAD112 was elevated in human umbilical vein endothelial cells transduced with scAAV2-CAD180 or scAAV2-CAD112, respectively, and both inhibited angiogenic activity in vitro. Intravitreal gene delivery of scAAV2-CAD180 or scAAV2-CAD112 significantly inhibited ischemia-induced retinal neovascularization in rat eyes (CAD180: 52.7% reduction; CAD112: 49.2% reduction) compared to scAAV2-mCherry, as measured in retinal flatmounts stained with isolectin B4. Moreover, the retinal structure and function were unaffected by scAAV2-CAD180 or scAAV2-CAD112, as measured by optical coherence tomography and electroretinography. Moreover, subretinal delivery of scAAV2-CAD180 or scAAV2-CAD112 significantly attenuated laser-induced choroidal neovascularization in mouse eyes compared to scAAV2-mCherry, as measured by fundus fluorescein angiography (CAD180: 62.4% reduction; CAD112: 57.5% reduction) and choroidal flatmounts (CAD180: 40.21% reduction; CAD112: 43.03% reduction). Gene delivery using scAAV2-CAD180 or scAAV2-CAD112 has significant potential as a therapeutic option for the management of ocular neovascularization.
Assuntos
Inibidores da Angiogênese/biossíntese , Calreticulina , Dependovirus , Retinopatia Diabética , Neovascularização Retiniana , Transdução Genética , Inibidores da Angiogênese/genética , Angiografia , Animais , Calreticulina/biossíntese , Calreticulina/genética , Retinopatia Diabética/diagnóstico por imagem , Retinopatia Diabética/genética , Retinopatia Diabética/metabolismo , Retinopatia Diabética/fisiopatologia , Eletrorretinografia , Feminino , Vetores Genéticos , Células HEK293 , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais da Veia Umbilical Humana/patologia , Humanos , Proteínas Luminescentes/biossíntese , Proteínas Luminescentes/genética , Degeneração Macular/diagnóstico por imagem , Degeneração Macular/genética , Degeneração Macular/metabolismo , Degeneração Macular/fisiopatologia , Camundongos , Ratos , Ratos Sprague-Dawley , Neovascularização Retiniana/diagnóstico por imagem , Neovascularização Retiniana/genética , Neovascularização Retiniana/metabolismo , Neovascularização Retiniana/fisiopatologia , Tomografia de Coerência Óptica , Proteína Vermelha FluorescenteRESUMO
NOTCH signalling is an evolutionarily conserved juxtacrine signalling pathway that is essential in development. Jagged1 (JAG1) and Delta-like ligand 4 (DLL4) are transmembrane NOTCH ligands that regulate angiogenesis by controlling endothelial cell (EC) differentiation, vascular development and maturation. In addition, DLL4 could bypass its canonical cell-cell contact-dependent signalling to influence NOTCH signalling and angiogenesis at a distance when it is packaged into extracellular vesicles (EVs). However, it is not clear whether JAG1 could also be packaged into EVs to influence NOTCH signalling and angiogenesis. In this work, we demonstrate that JAG1 is also packaged into EVs. We present evidence that JAG1-EVs inhibit NOTCH signalling and regulate EC behaviour and function. JAG1-EVs inhibited VEGF-induced HUVEC proliferation and migration in 2D culture condition and suppressed sprouting in a 3D microfluidic microenvironment. JAG1-EV treatment of HUVECs leads to a reduction of Notch1 intracellular domain (N1-ICD), and the proteasome and the intracellular domain of JAG1 (JAG1-ICD) are both required for this reduction to occur. These findings reveal a novel mechanism of JAG1 function in NOTCH signalling and ECs through EVs.
Assuntos
Inibidores da Angiogênese/metabolismo , Microambiente Celular/fisiologia , Vesículas Extracelulares/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Proteína Jagged-1/metabolismo , Neovascularização Fisiológica , Receptores Notch/metabolismo , Transdução de Sinais/fisiologia , Inibidores da Angiogênese/genética , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Vesículas Extracelulares/genética , Células HEK293/metabolismo , Células HEK293/fisiologia , Células Endoteliais da Veia Umbilical Humana/citologia , Humanos , Proteína Jagged-1/genética , Inibidores de Proteassoma/metabolismo , Domínios Proteicos , Receptores Notch/genética , Transdução de Sinais/genética , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
BACKGROUND: Long-term intraocular injections of vascular endothelial growth factor (VEGF)-neutralising proteins can preserve central vision in many patients with neovascular age-related macular degeneration. We tested the safety and tolerability of a single intravitreous injection of an AAV2 vector expressing the VEGF-neutralising protein sFLT01 in patients with advanced neovascular age-related macular degeneration. METHODS: This was a phase 1, open-label, dose-escalating study done at four outpatient retina clinics in the USA. Patients were assigned to each cohort in order of enrolment, with the first three patients being assigned to and completing the first cohort before filling positions in the following treatment groups. Patients aged 50 years or older with neovascular age-related macular degeneration and a baseline best-corrected visual acuity score of 20/100 or less in the study eye were enrolled in four dose-ranging cohorts (cohort 1, 2â×â108 vector genomes (vg); cohort 2, 2â×â109 vg; cohort 3, 6â×â109 vg; and cohort 4, 2â×â1010 vg, n=3 per cohort) and one maximum tolerated dose cohort (cohort 5, 2â×â1010 vg, n=7) and followed up for 52 weeks. The primary objective of the study was to assess the safety and tolerability of a single intravitreous injection of AAV2-sFLT01, through the measurement of eye-related adverse events. This trial is registered with ClinicalTrials.gov, number NCT01024998. FINDINGS: 19 patients with advanced neovascular age-related macular degeneration were enrolled in the study between May 18, 2010, and July 14, 2014. All patients completed the 52-week trial period. Two patients in cohort 4 (2 ×â1010 vg) experienced adverse events that were possibly study-drug related: pyrexia and intraocular inflammation that resolved with a topical steroid. Five of ten patients who received 2â×â1010 vg had aqueous humour concentrations of sFLT01 that peaked at 32·7-112·0 ng/mL (mean 73·7 ng/mL, SD 30·5) by week 26 with a slight decrease to a mean of 53·2 ng/mL at week 52 (SD 17·1). At baseline, four of these five patients were negative for anti-AAV2 serum antibodies and the fifth had a very low titre (1:100) of anti-AAV2 antibodies, whereas four of the five non-expressers of sFLT01 had titres of 1:400 or greater. In 11 of 19 patients with intraretinal or subretinal fluid at baseline judged to be reversible, six showed substantial fluid reduction and improvement in vision, whereas five showed no fluid reduction. One patient in cohort 5 showed a large decrease in vision between weeks 26 and 52 that was not thought to be vector-related. INTERPRETATION: Intravitreous injection of AAV2-sFLT01 seemed to be safe and well tolerated at all doses. Additional studies are needed to identify sources of variability in expression and anti-permeability activity, including the potential effect of baseline anti-AAV2 serum antibodies. FUNDING: Sanofi Genzyme, Framingham, MA, USA.
Assuntos
Terapia Genética/métodos , Degeneração Macular/terapia , Parvovirinae/genética , Proteínas Recombinantes de Fusão/genética , Idoso , Idoso de 80 Anos ou mais , Inibidores da Angiogênese/biossíntese , Inibidores da Angiogênese/genética , Neovascularização de Coroide/diagnóstico por imagem , Neovascularização de Coroide/fisiopatologia , Neovascularização de Coroide/terapia , Dependovirus , Feminino , Terapia Genética/efeitos adversos , Vetores Genéticos/administração & dosagem , Humanos , Injeções Intravítreas , Degeneração Macular/diagnóstico por imagem , Degeneração Macular/fisiopatologia , Masculino , Pessoa de Meia-Idade , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/biossíntese , Tomografia de Coerência Óptica , Acuidade VisualRESUMO
Cystatins are a family of cysteine protease inhibitors which are associated with a variety of physiological and pathological processes in vivo. In the present study, the cDNA sequence of a cystatin F homologue called Lm-cystatin F was cloned from the buccal glands of Lampetra morii. Although Lm-cystatin F shares a lower homology with cystatin superfamily members, it is also composed of a signal peptide and three highly conserved motifs, including the G in the N-terminal, QXVXG, as well as the PW in the C-terminal of the sequence. After sequence optimization and recombination, the recombinant protein was expressed as a soluble protein in Escherichia coli with a molecular weight of 19.85 kDa. Through affinity chromatography and mass spectrometry analysis, the purified protein was identified as a recombinant Lm-cystatin F (rLm-cystatin F). Additionally, rLm-cystatin F could inhibit the activity of papain. Based on MTT assay, rLm-cystatin F inhibited the proliferation of human umbilical vein endothelial cells (HUVECs) dose dependently with an IC50 of 5 µM. In vitro studies show that rLm-cystatin F suppressed the adhesion, migration, invasion, and tube formation of HUVECs, suggesting that rLm-cystatin F possesses anti-angiogenic activity, which provides information on the feeding mechanisms of Lampetra morii and insights into the application of rLm-cystatin F as a potential drug in the future.
Assuntos
Inibidores da Angiogênese/farmacologia , Cistatinas/farmacologia , Lampreias , Neovascularização Fisiológica/efeitos dos fármacos , Papaína/antagonistas & inibidores , Motivos de Aminoácidos , Sequência de Aminoácidos , Inibidores da Angiogênese/química , Inibidores da Angiogênese/genética , Inibidores da Angiogênese/isolamento & purificação , Animais , Proliferação de Células/efeitos dos fármacos , Cistatinas/química , Cistatinas/genética , Cistatinas/isolamento & purificação , DNA Complementar , Células Endoteliais da Veia Umbilical Humana , Humanos , Concentração Inibidora 50 , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacologia , Homologia de Sequência de AminoácidosRESUMO
Vascular Endothelial Growth Factor Receptor 2 (VEGFR-2) is the main mediator of angiogenic signaling in endothelial cells and a primary responder to VEGF. VEGF dependent VEGFR-2 activation regulates endothelial cell migration and proliferation, as well as vessel permeability. VEGF is presented as an antiparallel homodimer, and its binding to VEGFR-2 brings two receptors in close proximity. Downstream signaling is triggered by receptor dimerization, kinase activation, and receptor internalization. Our aim was to further investigate allosteric inhibition using binders targeting extracellular subdomains 4â»7 of VEGFR-2 as an alternative to existing anti-angiogenic therapies, which rely on neutralizing VEGF or blocking of the ligand-binding site on the receptor. We applied phage display technology to produce single chain antibody fragments (scFvs) targeting VEGFR-2. Selected antibody fragments were characterized using biophysical and biological assays. We characterized several antibody fragments, which exert their inhibitory effect of VEGFR-2 independent of ligand binding. These reagents led to rapid clearance of VEGFR-2 from the cell surface without kinase activation, followed by an increase in intracellular receptor-positive vesicles, suggesting receptor internalization. Our highly specific VEGFR-2 binders thus represent novel tools for anti-angiogenic therapy and diagnostic applications.
Assuntos
Inibidores da Angiogênese/farmacologia , Proteínas Recombinantes/farmacologia , Anticorpos de Cadeia Única/farmacologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Regulação Alostérica , Inibidores da Angiogênese/genética , Inibidores da Angiogênese/metabolismo , Animais , Aorta/citologia , Sítios de Ligação , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Técnicas de Visualização da Superfície Celular , Relação Dose-Resposta a Droga , Células HEK293 , Células Endoteliais da Veia Umbilical Humana , Humanos , Ligantes , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/metabolismo , Suínos , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/imunologiaRESUMO
Bispecific antibodies, which can bind to two different epitopes on the same or different antigens simultaneously, have recently emerged as attractive candidates for study in various diseases. Our present study successfully constructs and expresses a fully human, bispecific, single-chain diabody (BsDb) that can bind to vascular endothelial growth factor 165 (VEGF165) and programmed death-1 (PD-1) in Pichia pastoris. Under the optimal expression conditions (methanol concentration, 1%; pH, 4.0; inoculum density, OD600 = 4, and the induction time, 96 h), the maximum production level of this BsDb is achieved at approximately 20 mg/L. The recombinant BsDb is purified in one step using nickel-nitrilotriacetic acid (Ni-NTA) column chromatography with a purity of more than 95%. Indirect enzyme-linked immune sorbent assay (ELISA) and sandwich ELISA analyses show that purified BsDb can bind specifically to VEGF165 and PD-1 simultaneously with affinities of 124.78 nM and 25.07 nM, respectively. Additionally, the BsDb not only effectively inhibits VEGF165-stimulated proliferation, migration, and tube formation in primary human umbilical vein endothelial cells (HUVECs), but also significantly improves proliferation and INF-γ production of activated T cells by blocking PD-1/PD-L1 co-stimulation. Furthermore, the BsDb displays potent antitumor activity in mice bearing HT29 xenograft tumors by inhibiting tumor angiogenesis and activating immune responses in the tumor microenvironment. Based on these results, we have prepared a potential bispecific antibody drug that can co-target both VEGF165 and PD-1 for the first time. This work provides a stable foundation for the development of new strategies by the combination of an angiogenesis inhibition and immune checkpoint blockade for cancer therapy.
Assuntos
Anticorpos Biespecíficos/imunologia , Antineoplásicos Imunológicos/imunologia , Neoplasias/tratamento farmacológico , Receptor de Morte Celular Programada 1/imunologia , Anticorpos de Cadeia Única/imunologia , Fator A de Crescimento do Endotélio Vascular/imunologia , Inibidores da Angiogênese/genética , Inibidores da Angiogênese/imunologia , Inibidores da Angiogênese/farmacologia , Inibidores da Angiogênese/uso terapêutico , Animais , Anticorpos Biespecíficos/genética , Anticorpos Biespecíficos/farmacologia , Anticorpos Biespecíficos/uso terapêutico , Antineoplásicos Imunológicos/metabolismo , Antineoplásicos Imunológicos/farmacologia , Antineoplásicos Imunológicos/uso terapêutico , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Clonagem Molecular , Feminino , Expressão Gênica , Vetores Genéticos/genética , Células Endoteliais da Veia Umbilical Humana , Humanos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias/imunologia , Pichia/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/farmacologia , Proteínas Recombinantes/uso terapêutico , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/farmacologia , Anticorpos de Cadeia Única/uso terapêuticoRESUMO
Chronic kidney disease (CKD) is a major global health problem. Unless intensive intervention is initiated, some patients can rapidly progress to end-stage kidney disease. However, it is often difficult to predict renal outcomes using conventional laboratory tests in individuals with CKD. Therefore, many researchers have been searching for novel biomarkers to predict the progression of CKD. Angiogenesis is involved in physiological and pathological processes in the kidney and is regulated by the balance between a proangiogenic factor, vascular endothelial growth factor (VEGF)-A, and various endogenous antiangiogenic factors. In recent reports using genetically engineered mice, the roles of these antiangiogenic factors in the pathogenesis of kidney disease have become increasingly clear. In addition, recent clinical studies have demonstrated associations between circulating levels of antiangiogenic factors and renal dysfunction in CKD patients. In this review, we summarize recent advances in the study of representative endogenous antiangiogenic factors, including soluble fms-related tyrosine kinase 1, soluble endoglin, pigment epithelium-derived factor, VEGF-A165b, endostatin, and vasohibin-1, in associations with kidney diseases and discuss their predictive potentials as biomarkers of progression of CKD.
Assuntos
Inibidores da Angiogênese/genética , Proteínas do Olho/genética , Falência Renal Crônica/diagnóstico , Fatores de Crescimento Neural/genética , Insuficiência Renal Crônica/diagnóstico , Serpinas/genética , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/genética , Indutores da Angiogênese/química , Indutores da Angiogênese/metabolismo , Inibidores da Angiogênese/metabolismo , Animais , Biomarcadores/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Progressão da Doença , Endostatinas/genética , Endostatinas/metabolismo , Proteínas do Olho/metabolismo , Regulação da Expressão Gênica , Humanos , Falência Renal Crônica/etiologia , Falência Renal Crônica/genética , Falência Renal Crônica/patologia , Camundongos , Fatores de Crescimento Neural/metabolismo , Insuficiência Renal Crônica/complicações , Insuficiência Renal Crônica/genética , Insuficiência Renal Crônica/patologia , Serpinas/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
Neoangiogenesis is the main pathogenic event involved in a variety of retinal diseases. It has been recently demonstrated that inhibiting the urokinase-type plasminogen activator receptor (uPAR) results in reduced angiogenesis in a mouse model of oxygen-induced retinopathy (OIR), establishing uPAR as a therapeutic target in proliferative retinopathies. Here, we evaluated in cultured human retinal endothelial cells (HRECs) and in OIR mice the potential of a specific antisense oligodeoxyribonucleotide (ASO) in blocking the synthesis of uPAR and in providing antiangiogenic effects. uPAR expression in HRECs was inhibited by lipofection with the phosphorotioated 5'-CGGCGGGTGACCCATGTG-3' ASO-uPAR, complementary to the initial translation site of uPAR mRNA. Inhibition of uPAR expression via ASO-uPAR was evaluated in HRECs by analyzing VEGF-induced tube formation and migration. In addition, the well-established and reproducible murine OIR model was used to induce retinal neovascularization in vivo. OIR mice were injected intraperitoneally with ASO-uPAR and retinopathy was evaluated considering the extent of the avascular area in the central retina and neovascular tuft formation. The ASO-uPAR specifically decreased uPAR mRNA and protein levels in HRECs and mitigated VEGF-induced tube formation and cell migration. Noteworthy, in OIR mice ASO-uPAR administration reduced both the avascular area and the formation of neovascular tufts. In conclusion, although the extrapolation of these experimental findings to the clinic is not straightforward, ASO-uPAR may be considered a potential therapeutic tool for treatment of proliferative retinal diseases.
Assuntos
Inibidores da Angiogênese/uso terapêutico , Oligodesoxirribonucleotídeos Antissenso/uso terapêutico , Receptores de Ativador de Plasminogênio Tipo Uroquinase/genética , Retina/patologia , Neovascularização Retiniana/genética , Neovascularização Retiniana/terapia , Inibidores da Angiogênese/genética , Animais , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Modelos Animais de Doenças , Terapia Genética , Humanos , Camundongos , Oligodesoxirribonucleotídeos Antissenso/genética , RNA Mensageiro/genética , Receptores de Ativador de Plasminogênio Tipo Uroquinase/análise , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Retina/citologia , Retina/metabolismo , Neovascularização Retiniana/metabolismo , Neovascularização Retiniana/patologia , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Hepatocellular carcinoma (HCC) is a hypervascular cancer without effective treatment. Here we report that polypeptide of NC1 domain of type VIII collagen (Vastatin) is an endogenous polypeptide expressed in normal liver tissue but lost in the liver of most HCC patients (73.1%). Its expression level is negatively associated with tumor size (P = 0.035) and metastasis (P = 0.016) in HCC patients. To evaluate its potential use as a therapeutic, we constructed a recombinant adeno-associated virus carrying Vastatin (rAAV-Vastatin) to treat HCC in an orthotopic Buffalo rat model. rAAV-Vastatin treatment significantly prolonged the median survival, inhibited tumor growth, and completely prevented metastasis in HCC-bearing rats by decreasing microvessel density and increasing tumor necrosis. No detectable toxicity in nontumor-bearing mice was observed. To investigate its molecular mechanisms, we performed DNA microarray, western blotting assays, and bioinformatic analysis to determine its effect on global gene expression patterns and signal transduction pathways. Our results indicated that rAAV-Vastatin significantly reduced the expressions of Pck1, JAG2, and c-Fos, thus inhibiting the cellular metabolism, Notch and AP-1 signaling pathways, respectively. Hence, we demonstrated for the first time that Vastatin is a novel, safe, and effective antiangiogenic therapeutic and a potential biomarker for HCC.
Assuntos
Inibidores da Angiogênese/genética , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Colágeno Tipo VIII/genética , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Adulto , Idoso , Inibidores da Angiogênese/metabolismo , Animais , Carcinoma Hepatocelular/metabolismo , Linhagem Celular Tumoral , Colágeno Tipo VIII/metabolismo , Dependovirus/genética , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Feminino , Expressão Gênica , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Humanos , Neoplasias Hepáticas/metabolismo , Masculino , Pessoa de Meia-Idade , Necrose , Gradação de Tumores , Metástase Neoplásica , Neovascularização Patológica/genética , Ratos , Receptores Notch/metabolismo , Transdução de Sinais , Fator de Transcrição AP-1/metabolismo , Transdução Genética , Carga Tumoral , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Angiogenesis, one of the hallmarks of cancers, has become an attractive target for cancer therapy since decades ago. It is broadly thought that upregulation of angiogenesis is involved in tumor progression and metastasis. Though tumor vessels are tortuous, disorganized, and leaky, they deliver oxygen and nutrients for tumor development. Based on this knowledge, many kinds of drugs targeting angiogenesis pathways have been developed, such as bevacizumab. However, the clinical outcomes of anti-angiogenesis therapies are moderate in metastatic breast cancer as well as in metastatic colorectal cancer and non-small cell lung cancer, even combined with traditional chemotherapy. In this chapter, the morphologic angiogenesis patterns and the key molecular pathways regulating angiogenesis are elaborated. The FDA-approved anti-angiogenesis drugs and current challenges of anti-angiogenesis therapy are described. The strategies to overcome the barriers will also be elucidated.
Assuntos
Inibidores da Angiogênese/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Metástase Neoplásica/tratamento farmacológico , Neovascularização Patológica/tratamento farmacológico , Inibidores da Angiogênese/genética , Anticorpos Monoclonais/uso terapêutico , Bevacizumab/genética , Bevacizumab/uso terapêutico , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Feminino , Humanos , Imunoterapia/tendências , Metástase Neoplásica/genética , Metástase Neoplásica/patologia , Neovascularização Patológica/genética , Neovascularização Patológica/patologiaRESUMO
MicroRNAs (miRNAs) modulate complex physiological and pathological processes, including the regulation of angiogenesis. Our previous study reported that bone marrow-derived mesenchymal stem cells (MSCs) are recruited into choroidal neovascularization lesions. miRNA-195 is highly expressed in MSCs, but its function remains unknown. In the present study, miR-195a-3p abundance was significantly decreased in hypoxia-treated murine MSCs; on the other hand, its overexpression reduced MSC proliferation and migration while increasing the activation of anti-angiogenic factor pigment epithelium-derived factor (PEDF). We further discovered that matrix metalloproteinase 2 (Mmp2) transcript is a target of miR-195a-3p, and that silencing Mmp2 phenocopied the reduced proliferation and migration of MSCs. The therapeutic potential of miR-195a-3p as an angiogenesis inhibitor was also demonstrated in a laser-induced choroidal neovascularization mouse model. These findings collectively indicate that miR-195a-3p is a negative modulator of angiogenesis, and could be used as an angiogenesis inhibitor. Mol. Reprod. Dev. 83: 413-423, 2016. © 2016 Wiley Periodicals, Inc.
Assuntos
Inibidores da Angiogênese/metabolismo , Neovascularização de Coroide/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Células-Tronco Mesenquimais/metabolismo , MicroRNAs/metabolismo , Inibidores da Angiogênese/genética , Animais , Células da Medula Óssea/patologia , Neovascularização de Coroide/patologia , Modelos Animais de Doenças , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Feminino , Células-Tronco Mesenquimais/patologia , Camundongos , MicroRNAs/genética , Fatores de Crescimento Neural/genética , Fatores de Crescimento Neural/metabolismo , Serpinas/genética , Serpinas/metabolismoRESUMO
BACKGROUND This study investigated how miR-21 expression is reflected in acute myocardial infarction and explored the role of miR-21 and the PTEN/VEGF signaling pathway in cardiac microvascular endothelial cells. MATERIAL AND METHODS We used an in vivo LAD rat model to simulate acute myocardial infarction. MiR-21 mimics and miR-21 inhibitors were injected and transfected into model rats in order to alter miR-21 expression. Cardiac functions were evaluated using echocardiographic measurement, ELISA, and Masson staining. In addition, lenti-PTEN and VEGF siRNA were transfected into CMEC cells using standard procedures for assessing the effect of PTEN and VEGE on cell proliferation, apoptosis, and angiogenesis. MiR-21, PTEN, and VEGF expressions were examined by RT-PCR and Western blot. The relationship between miR-21 and PTEN was determined by the luciferase activity assay. RESULTS We demonstrated that miR-21 bonded with the 3'-UTR of PTEN and suppressed PTEN expressions. Established models significantly induced cardiac infarct volume and endothelial injury marker expressions as well as miR-21 and PTEN expressions (P<0.05). MiR-21 mimics exhibited significantly protective effects since they down-regulated both infarction size and injury marker expressions by increasing VEGF expression and inhibiting PTEN expression (P<0.05). In addition, results from in vitro research show that lenti-PTEN and VEGF siRNA can notably antagonize the effect of miR-21 on cell proliferation, apoptosis, and angiogenesis (P<0.05). CONCLUSIONS MiR-21 exerts protective effects on endothelial injury through the PTEN/VEGF pathway after acute myocardial infarction.
Assuntos
MicroRNAs/metabolismo , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Regiões 3' não Traduzidas , Inibidores da Angiogênese/genética , Inibidores da Angiogênese/metabolismo , Animais , Apoptose/genética , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Células-Tronco Mesenquimais , MicroRNAs/biossíntese , MicroRNAs/genética , Microvasos/patologia , Infarto do Miocárdio/genética , Neovascularização Patológica/genética , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Tensinas/metabolismo , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
In the course of conducting a series of studies whose goal was to discover novel endogenous angiogenesis inhibitors, we have purified matrilin-1 (MATN-1) and have demonstrated, for the first time, that it inhibits neovascularization both in vitro and in vivo. Proteins were extracted from cartilage using a 2 m NaCl, 0.01 m HEPES buffer at 4 °C, followed by concentration of the extract. The concentrate was fractionated by size exclusion chromatography, and fractions were then screened for their ability to inhibit capillary endothelial cell (EC) proliferation in vitro. Fractions containing EC inhibitory activity were pooled and further purified by cation exchange chromatography. The resulting fractions from this step were then screened to isolate the antiangiogenic activity in vitro. This activity was identified by tandem mass spectrometry as being MATN-1. Human MATN-1 was cloned and expressed in Pichia pastoris and purified to homogeneity. Purified recombinant MATN-1, along with purified native protein, was shown to inhibit angiogenesis in vivo using the chick chorioallantoic membrane assay by the inhibition of capillary EC proliferation and migration. Finally, using a MATN-1-deficient mouse, we showed that angiogenesis during fracture healing was significantly higher in MATN-1(-/-) mice compared with the wild type mice as demonstrated by in vivo imaging and by elevated expression of angiogenesis markers including PECAM1, VEGFR, and VE-cadherin.
Assuntos
Inibidores da Angiogênese/metabolismo , Proteínas Matrilinas/metabolismo , Neovascularização Fisiológica , Sequência de Aminoácidos , Inibidores da Angiogênese/química , Inibidores da Angiogênese/genética , Inibidores da Angiogênese/farmacologia , Animais , Bovinos , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Galinhas , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Fraturas Ósseas/metabolismo , Fraturas Ósseas/fisiopatologia , Técnicas de Inativação de Genes , Humanos , Masculino , Proteínas Matrilinas/química , Proteínas Matrilinas/genética , Proteínas Matrilinas/farmacologia , Camundongos , Dados de Sequência Molecular , Neovascularização Fisiológica/efeitos dos fármacos , Tíbia/lesões , CicatrizaçãoRESUMO
The soluble vascular endothelial growth factor (VEGF) receptor 1 (sFLT1) has been tested in both animals and humans for anti-angiogenic therapies, for example, age-related macular degeneration. We hypothesized that adeno-associated viral vector (AAV)-mediated sFLT1 expression could be used to inhibit abnormal brain angiogenesis. We tested the anti-angiogenic effect of sFLT1 and the feasibility of using AAV serotype 9 to deliver sFLT1 through intravenous injection (IV) to the brain angiogenic region. AAVs were packaged in AAV serotypes 1 and 2 (stereotactic injection) and 9 (IV injection). Brain angiogenesis was induced in adult mice through stereotactic injection of AAV1-VEGF. AAV2-sFLT02 containing sFLT1 VEGF-binding domain (domain 2) was injected into the brain angiogenic region, and AAV9-sFLT1 was injected into the jugular vein at the time of or 4 weeks after AAV1-VEGF injection. We showed that AAV2-sFLT02 inhibited brain angiogenesis at both time points. IV injection of AAV9-sFLT1 inhibited angiogenesis only when the vector was injected 4 weeks after angiogenic induction. Neither lymphocyte infiltration nor neuron loss was observed in AAV9-sFLT1-treated mice. Our data show that systemically delivered AAV9-sFLT1 inhibits angiogenesis in the mouse brain, which could be utilized to treat brain angiogenic diseases such as brain arteriovenous malformation.
Assuntos
Inibidores da Angiogênese/genética , Encéfalo/irrigação sanguínea , Dependovirus/genética , Técnicas de Transferência de Genes , Vetores Genéticos/administração & dosagem , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/genética , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Encéfalo/virologia , Terapia Genética , Vetores Genéticos/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neovascularização Patológica/genética , Neovascularização Patológica/metabolismo , Neovascularização Patológica/terapia , Distribuição Aleatória , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/biossínteseRESUMO
Various ways to inhibit vascular endothelial growth factor (VEGF), a key facilitator in tumor angiogenesis, are being developed to treat cancer. The soluble VEGF decoy receptor (FP3), due to its high affinity to VEGF, is a highly effective and promising strategy to disrupt VEGF signaling pathway. Despite potential advantage and potent therapeutic efficacy, its employment has been limited by very poor in vivo pharmacokinetic properties. To address this challenge, we designed a novel oncolytic adenovirus (Ad) expressing FP3 (RdB/FP3). To demonstrate the VEGF-specific nature of RdB/FP3, replication-incompetent Ad expressing FP3 (dE1/FP3) was also generated. dE1/FP3 was highly effective in reducing VEGF expression and functionally elicited an antiangiogeneic effect. Furthermore, RdB/FP3 exhibited a potent antitumor effect compared with RdB or recombinant FP3. Consistent with these data, RdB/FP3 was shown to greatly decrease VEGF expression level and vessel density and increase apoptosis in both tumor endothelial and tumor cells, verifying potent suppressive effects of RdB/FP3 on VEGF-mediated tumor angiogenesis in vivo. Importantly, the therapeutic mechanism of antitumor effect mediated by RdB/FP3 is associated with prolonged VEGF silencing efficacy and enhanced oncolysis via cancer cell-specific replication of oncolytic Ad. Taken together, RdB/FP3 provides a new promising therapeutic approach in the treatment of cancer and angiogenesis-related diseases.