RESUMEN
The formation of new vessels in the tumor, termed angiogenesis, is essential for primary tumor growth and facilitates tumor invasion and metastasis. Hypoxia has been described as one trigger of angiogenesis. Indeed, hypoxia, which is characterized by areas of low oxygen levels, is a hallmark of solid tumors arising from an imbalance between oxygen delivery and consumption. Hypoxic conditions have profound effects on the different components of the tumoral environment. For example, hypoxia is able to activate endothelial cells, leading to angiogenesis but also thereby initiating a cascade of reactions involving neutrophils, smooth muscle cells, and fibroblasts. In addition, hypoxia directly regulates the expression of many genes for which the role and the importance in the tumoral environment remain to be completely elucidated. In this study, we used a method to selectively label sialoglycoproteins to identify new membrane and secreted proteins involved in the adaptative process of endothelial cells by mass spectrometry-based proteomics. We used an in vitro assay under hypoxic condition to observe an increase of protein expression or modifications of glycosylation. Then the function of the identified proteins was assessed in a vasculogenesis assay in vivo by using a morpholino strategy in zebrafish. First, our approach was validated by the identification of sialoglycoproteins such as CD105, neuropilin-1, and CLEC14A, which have already been described as playing key roles in angiogenesis. Second, we identified several new proteins regulated by hypoxia and demonstrated for the first time the pivotal role of GLUT-1, TMEM16F, and SDF4 in angiogenesis.
Asunto(s)
Neovascularización Fisiológica , Procesamiento Proteico-Postraduccional , Sialoglicoproteínas/metabolismo , Adaptación Fisiológica , Animales , Anoctaminas , Antígenos CD/genética , Antígenos CD/metabolismo , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/metabolismo , Hipoxia de la Célula , Endoglina , Transportador de Glucosa de Tipo 1/genética , Transportador de Glucosa de Tipo 1/metabolismo , Glicoproteínas/genética , Glicoproteínas/metabolismo , Glicosilación , Células Endoteliales de la Vena Umbilical Humana , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Neuropilina-1/genética , Neuropilina-1/metabolismo , Proteínas de Transferencia de Fosfolípidos/genética , Proteínas de Transferencia de Fosfolípidos/metabolismo , Proteoma/química , Proteoma/metabolismo , Proteómica/métodos , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Sialoglicoproteínas/genética , Pez CebraRESUMEN
Tumor angiogenesis is accompanied by vasculogenesis, which is involved in the differentiation and mobilization of human bone marrow cells. In order to further characterize the role of vasculogenesis in the tumor growth process, the effects of FGF2 on the differentiation of human bone marrow AC133(+) cells (BM-AC133(+)) into vascular precursors were studied in vitro. FGF2, like VEGFA, induced progenitor cell differentiation into cell types with endothelial cell characteristics. SSR128129E, a newly discovered specific FGFR antagonist acting by allosteric interaction with FGFR, abrogated FGF2-induced endothelial cell differentiation, showing that FGFR signaling is essential during this process. To assess the involvement of the FGF/FRGR signaling in vivo, the pre-clinical model of Lewis lung carcinoma (LL2) in mice was used. Subcutaneous injection of LL2 cells into mice induced an increase of circulating EPCs from peripheral blood associated with tumor growth and an increase of intra-tumoral vascular index. Treatment with the FGFR antagonist SSR128129E strongly decreased LL2 tumor growth as well as the intra-tumoral vascular index (41% and 50% decrease vs. vehicle-treated mice respectively, P < 0.01). Interestingly, SSR128129E treatment significantly decreased the number of circulating EPCs from the peripheral blood (53% inhibition vs. vehicle-treated mice, P < 0.01). These results demonstrate for the first time that the blockade of the FGF/FGFR pathway by SSR128129E reduces EPC recruitment during angiogenesis-dependent tumor growth. In this context, circulating EPCs could be a reliable surrogate marker for tumor growth and angiogenic activity.
Asunto(s)
Carcinoma Pulmonar de Lewis/irrigación sanguínea , Células Madre Hematopoyéticas/citología , Indolizinas/farmacología , Neovascularización Patológica/patología , Receptores de Factores de Crecimiento de Fibroblastos/antagonistas & inhibidores , ortoaminobenzoatos/farmacología , Antígeno AC133 , Animales , Antígenos CD/biosíntesis , Células de la Médula Ósea/metabolismo , Adhesión Celular , Diferenciación Celular/efectos de los fármacos , Línea Celular , Movimiento Celular , Células Endoteliales/citología , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Glicoproteínas/biosíntesis , Humanos , Ratones , Ratones Endogámicos C57BL , Péptidos , ARN Mensajero/biosíntesis , Receptores de Factores de Crecimiento de Fibroblastos/genética , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Transducción de SeñalRESUMEN
VEGF-B, a homolog of VEGF discovered a long time ago, has not been considered an important target in antiangiogenic therapy. Instead, it has received little attention from the field. In this study, using different animal models and multiple types of vascular cells, we revealed that although VEGF-B is dispensable for blood vessel growth, it is critical for their survival. Importantly, the survival effect of VEGF-B is not only on vascular endothelial cells, but also on pericytes, smooth muscle cells, and vascular stem/progenitor cells. In vivo, VEGF-B targeting inhibited both choroidal and retinal neovascularization. Mechanistically, we found that the vascular survival effect of VEGF-B is achieved by regulating the expression of many vascular prosurvival genes via both NP-1 and VEGFR-1. Our work thus indicates that the function of VEGF-B in the vascular system is to act as a "survival," rather than an "angiogenic" factor and that VEGF-B inhibition may offer new therapeutic opportunities to treat neovascular diseases.
Asunto(s)
Neovascularización Patológica , Factor B de Crecimiento Endotelial Vascular/metabolismo , Animales , Supervivencia Celular , Células Cultivadas , Modelos Animales de Enfermedad , Perfilación de la Expresión Génica , Genoma , Miembro Posterior/irrigación sanguínea , Isquemia/genética , Isquemia/metabolismo , Ratones , Ratones Noqueados , Ratas , Retina/metabolismo , Células Madre/citología , Células Madre/metabolismo , Regulación hacia Arriba , Factor B de Crecimiento Endotelial Vascular/deficiencia , Factor B de Crecimiento Endotelial Vascular/genéticaRESUMEN
The receptor tyrosine kinase VEGFR-3 plays a crucial role in cancer-induced angiogenesis and lymphangiogenesis, promoting tumor development and metastasis. Here, we report the novel VEGFR-3 inhibitor EVT801 that presents a more selective and less toxic profile than two major inhibitors of VEGFRs (i.e., sorafenib and pazopanib). As monotherapy, EVT801 showed a potent antitumor effect in VEGFR-3-positive tumors, and in tumors with VEGFR-3-positive microenvironments. EVT801 suppressed VEGF-C-induced human endothelial cell proliferation in vitro and tumor (lymph)angiogenesis in different tumor mouse models. In addition to reduced tumor growth, EVT801 decreased tumor hypoxia, favored sustained tumor blood vessel homogenization (i.e., leaving fewer and overall larger vessels), and reduced important immunosuppressive cytokines (CCL4, CCL5) and myeloid-derived suppressor cells (MDSC) in circulation. Furthermore, in carcinoma mouse models, the combination of EVT801 with immune checkpoint therapy (ICT) yielded superior outcomes to either single treatment. Moreover, tumor growth inhibition was inversely correlated with levels of CCL4, CCL5, and MDSCs after treatment with EVT801, either alone or combined with ICT. Taken together, EVT801 represents a promising anti(lymph)angiogenic drug for improving ICT response rates in patients with VEGFR-3 positive tumors. Significance: The VEGFR-3 inhibitor EVT801 demonstrates superior selectivity and toxicity profile than other VEGFR-3 tyrosine kinase inhibitors. EVT801 showed potent antitumor effects in VEGFR-3-positive tumors, and tumors with VEGFR-3-positive microenvironments through blood vessel homogenization, and reduction of tumor hypoxia and limited immunosuppression. EVT801 increases immune checkpoint inhibitors' antitumor effects.
Asunto(s)
Neoplasias , Receptor 3 de Factores de Crecimiento Endotelial Vascular , Humanos , Ratones , Animales , Receptor 3 de Factores de Crecimiento Endotelial Vascular/uso terapéutico , Neovascularización Patológica/tratamiento farmacológico , Neoplasias/tratamiento farmacológico , Inhibidores de la Angiogénesis/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Inmunoterapia , Microambiente TumoralRESUMEN
The angiogenic mechanism and therapeutic potential of PDGF-CC, a recently discovered member of the VEGF/PDGF superfamily, remain incompletely characterized. Here we report that PDGF-CC mobilized endothelial progenitor cells in ischemic conditions; induced differentiation of bone marrow cells into ECs; and stimulated migration of ECs. Furthermore, PDGF-CC induced the differentiation of bone marrow cells into smooth muscle cells and stimulated their growth during vessel sprouting. Moreover, delivery of PDGF-CC enhanced postischemic revascularization of the heart and limb. Modulating the activity of PDGF-CC may provide novel opportunities for treating ischemic diseases.
Asunto(s)
Células Endoteliales/citología , Células Endoteliales/efectos de los fármacos , Isquemia/tratamiento farmacológico , Isquemia/patología , Neovascularización Fisiológica/efectos de los fármacos , Factor de Crecimiento Derivado de Plaquetas/farmacología , Células Madre/efectos de los fármacos , Animales , Diferenciación Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Vasos Coronarios/citología , Vasos Coronarios/efectos de los fármacos , Miembro Posterior/irrigación sanguínea , Miembro Posterior/efectos de los fármacos , Humanos , Isquemia/inducido químicamente , Isquemia/metabolismo , Linfocinas , Ratones , Microcirculación/efectos de los fármacos , Miocardio/metabolismo , Miocardio/patología , Fosfotirosina/metabolismo , Receptores del Factor de Crecimiento Derivado de Plaquetas/metabolismo , Transducción de Señal , Células Madre/citologíaRESUMEN
In this review, we summarize the results of a number of our recent in vitro and in vivo experiments demonstrating that, in addition to the immunoregulatory functions, soluble HLA-G molecules also affect endothelial cell activity. We have found that soluble HLA-G1 (also designated HLA-G5) inhibits endothelial cell proliferation, migration and tubule formation, and this occurred through binding to the CD160 receptor and via an apoptotic pathway. Moreover, we have demonstrated that soluble HLA-G1 blocks in vivo rabbit corneal neoangiogenesis. Although it cannot be excluded that other soluble HLA class I molecules may have similar effects, as soluble forms of HLA-G are being produced by trophoblast cells at the maternal-fetal interface during early gestation, we discuss how such anti-angiogenic properties of soluble HLA-G1 may locally influence uterine vascular remodeling.
Asunto(s)
Antígenos CD/metabolismo , Antígenos HLA/metabolismo , Antígenos de Histocompatibilidad Clase I/metabolismo , Neovascularización Fisiológica , Receptores Inmunológicos/metabolismo , Moduladores de la Angiogénesis/metabolismo , Animales , Células Endoteliales/fisiología , Femenino , Proteínas Ligadas a GPI , Antígenos HLA-G , Humanos , Placenta/irrigación sanguínea , Placenta/inmunología , Embarazo , Trofoblastos/inmunología , Útero/irrigación sanguíneaRESUMEN
Angiogenesis is the biological process of generating new capillary blood vessels. It is a fundamental component of a number of normal (reproduction and wound healing) and pathological processes (diabetic retinopathy, rheumatoid arthritis, tumor growth, and metastasis). In vitro angiogenesis assays provide a platform for evaluating the effects of pro- or antiangiogenic compounds. One of the most informative assays is the endothelial cells capillary tube formation assay performed on a biological matrix. This assay is based on quantification of the stimulatory and inhibitory effects of various agents, which is estimated through the measurement of the pseudo-tubules network length. This standard measurement is usually carried out manually by trained operators but requires time, attention, and dedication to achieve a reasonable degree of accuracy. Moreover, the screening is operator dependent. In this article, we propose an automated procedure to evaluate the pseudo-tubule network lengths. We propose a series of image analysis procedures developed using a freely available image analysis software library. More than 800 images from 12 experiments were analyzed automatically and manually, and their results were compared to improve and validate the proposed image analysis procedure. The resulting image analysis software is currently running on a dedicated server, with comparable accuracy to manual measurements. Using this new automated procedure, we are able to treat 540 images, or three complete assays per hour.
Asunto(s)
Técnicas Citológicas/métodos , Células Endoteliales/efectos de los fármacos , Procesamiento de Imagen Asistido por Computador/métodos , Neovascularización Fisiológica/efectos de los fármacos , Humanos , Programas InformáticosRESUMEN
Receptor tyrosine kinases (RTK) are targets for anticancer drug development. To date, only RTK inhibitors that block orthosteric binding of ligands and substrates have been developed. Here, we report the pharmacologic characterization of the chemical SSR128129E (SSR), which inhibits fibroblast growth factor receptor (FGFR) signaling by binding to the extracellular FGFR domain without affecting orthosteric FGF binding. SSR exhibits allosteric properties, including probe dependence, signaling bias, and ceiling effects. Inhibition by SSR is highly conserved throughout the animal kingdom. Oral delivery of SSR inhibits arthritis and tumors that are relatively refractory to anti-vascular endothelial growth factor receptor-2 antibodies. Thus, orally-active extracellularly acting small-molecule modulators of RTKs with allosteric properties can be developed and may offer opportunities to improve anticancer treatment.
Asunto(s)
Inhibidores de Proteínas Quinasas/farmacología , Receptores de Factores de Crecimiento de Fibroblastos/antagonistas & inhibidores , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Bibliotecas de Moléculas Pequeñas/farmacología , Regulación Alostérica , Animales , Anticuerpos Monoclonales/farmacología , Artritis Experimental/tratamiento farmacológico , Resorción Ósea/tratamiento farmacológico , Carcinoma Pulmonar de Lewis/tratamiento farmacológico , Carcinoma Pulmonar de Lewis/metabolismo , Carcinoma Pulmonar de Lewis/patología , Factores de Crecimiento de Fibroblastos/antagonistas & inhibidores , Factores de Crecimiento de Fibroblastos/metabolismo , Células HEK293 , Células Endoteliales de la Vena Umbilical Humana/citología , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Ratones , Neovascularización Patológica/tratamiento farmacológico , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Fosforilación/efectos de los fármacos , Inhibidores de Proteínas Quinasas/metabolismo , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Transducción de Señal , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Neuropilin 2 (NRP2) is a receptor for the vascular endothelial growth factor (VEGF) and the semaphorin (SEMA) families, 2 unrelated ligand families involved in angiogenesis and neuronal guidance. NRP2 specifically binds VEGF-A and VEGF-C, although the biological relevance of these interactions in human endothelial cells is poorly understood. In this study, we show that both VEGF-A and VEGF-C induce the interaction of NRP2 with VEGFR-2. This interaction correlated with an enhancement of the VEGFR-2 phosphorylation threshold. Overexpression of NRP2 in primary human endothelial cells promoted cell survival induced by VEGF-A and VEGF-C. In contrast, SEMA3F, another ligand for NRP2, was able to inhibit human endothelial cell survival and migration induced by VEGF-A and VEGF-C. Moreover, a siRNA targeting specifically NRP2 was a potent inhibitor of human endothelial cell migration induced by VEGF-A and VEGF-C. Thus, our data indicate that NRP2 acts as a coreceptor that enhances human endothelial cell biological responses induced by VEGF-A and VEGF-C.
Asunto(s)
Movimiento Celular/fisiología , Células Endoteliales/fisiología , Neuropilina-2/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Receptor 3 de Factores de Crecimiento Endotelial Vascular/metabolismo , Línea Celular , Movimiento Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Expresión Génica , Humanos , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/farmacología , Proteínas del Tejido Nervioso/metabolismo , Proteínas del Tejido Nervioso/farmacología , Neuropilina-2/antagonistas & inhibidores , Unión Proteica/efectos de los fármacos , Unión Proteica/fisiología , ARN Interferente Pequeño/farmacología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Factor A de Crecimiento Endotelial Vascular/metabolismo , Factor A de Crecimiento Endotelial Vascular/farmacología , Factor C de Crecimiento Endotelial Vascular/metabolismo , Factor C de Crecimiento Endotelial Vascular/farmacologíaRESUMEN
HLA-G is a major histocompatibility complex class Ib molecule whose constitutive tissue distribution is restricted mainly to trophoblast cells at the maternal-fetal interface during pregnancy. In this study, we demonstrated the ability of the soluble HLA-G1 (sHLA-G1) isoform to inhibit fibroblast growth factor-2 (FGF2)-induced capillary-like tubule formation. Using a rabbit corneal neovascularization model, we further showed that sHLA-G1 inhibits FGF2-induced angiogenesis in vivo. We also demonstrated that sHLA-G1 induces endothelial cell apoptosis through binding to BY55/CD160, a glycosylphosphatidylinositolanchored receptor expressed by endothelial cells. Furthermore, we showed that the specific CL1-R2 anti-CD160 monoclonal antibody mimics sHLA-G1-mediated inhibition of endothelial cell tube formation and induction of apoptosis. Thus, the engagement of CD160 in endothelial cells may be essential for the inhibition of angiogenesis. sHLA-G1/CD160-mediated antiangiogenic property may participate in the vascular remodeling of maternal spiral arteries during pregnancy, and, given that we found that CD160 is strongly expressed in the vasculature of a murine tumor, it offers an attractive therapeutic target for preventing pathologic neovascularization.
Asunto(s)
Antígenos CD/metabolismo , Células Endoteliales/efectos de los fármacos , Antígenos HLA/metabolismo , Antígenos HLA/farmacología , Antígenos de Histocompatibilidad Clase I/metabolismo , Antígenos de Histocompatibilidad Clase I/farmacología , Neovascularización Fisiológica/efectos de los fármacos , Receptores Inmunológicos/metabolismo , Secuencia de Aminoácidos , Animales , Antígenos CD/genética , Apoptosis/efectos de los fármacos , Apoptosis/inmunología , Apoptosis/fisiología , Células Cultivadas , Córnea/irrigación sanguínea , Córnea/efectos de los fármacos , Células Endoteliales/citología , Células Endoteliales/inmunología , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Proteínas Ligadas a GPI , Antígenos HLA-G , Humanos , Técnicas In Vitro , Ratones , Datos de Secuencia Molecular , Conejos , Receptores Inmunológicos/genética , SolubilidadRESUMEN
Tumor growth and metastasis require the generation of new blood vessels, a process known as neo-angiogenesis. Recent studies have indicated that early tumor vascularization is characterized by the differentiation and mobilization of human bone marrow cells. Vascular endothelial growth factor-A (VEGF-A) is one of the growth factors, which enhances their differentiation into endothelial cells, but little is known about the implication of the VEGF-receptor tyrosine kinases and about the implication of the VEGF-R co-receptor, neuropilin-1, in this process. In this context, the identification of the molecular pathways that support the proliferation and differentiation of vascular stem and progenitor cells was investigated in order to define the pharmaceutical targets involved in tissue vascularization associated with this process. For this purpose, an in vitro model of differentiation of human bone marrow AC133+ (BM-AC133+) cells into vascular precursors was used. In this work, we have demonstrated for the first time that the effect of VEGF-A on BM-AC133+ cells relies on an early action of VEGF-A on the expression of its tyrosine kinase receptors followed by an activation of a VEGF-R2/neuropilin-1-dependent signaling pathway. This signaling promotes the differentiation of BM-AC133+ cells into endothelial precursor cells, followed by the proliferation of these differentiated cells. Altogether, these results strongly suggest that VEGF inhibitors, acting at the level of VEGF-R2 and/or neuropilin-1, by inhibiting differentiation and proliferation of these cells, could be potentially active compounds to prevent progenitor cells to be involved in tumor angiogenesis leading to tumor growth.
Asunto(s)
Células de la Médula Ósea/fisiología , Diferenciación Celular , Células Madre Hematopoyéticas/fisiología , Neuropilina-1/metabolismo , Receptores de Factores de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Factor A de Crecimiento Endotelial Vascular/metabolismo , Adhesión Celular , División Celular , Movimiento Celular , Células Cultivadas , Endotelio Vascular/citología , Endotelio Vascular/fisiología , Humanos , Neovascularización Patológica/prevención & control , Molécula-1 de Adhesión Celular Endotelial de Plaqueta , Receptores de Factores de Crecimiento Endotelial Vascular/metabolismo , Transducción de Señal , Factores de TiempoRESUMEN
VEGFR-3 is essential for vascular development and maintenance of lymphatic vessel's integrity. Little is known about its cooperative effect with other receptors of the same family. Contrary to VEGFR-2, stimulation of VEGFR-3 by VEGF-C and -D failed to enhance its phosphorylation either in HEK293T or in PAE cells. These ligands were unable to induce angiogenesis of PAEC expressing VEGFR-3 alone. In the presence of VEGFR-2, VEGF-C and -D induced heterodimerization of VEGFR-3 with VEGFR-2. This heterodimerization was associated with enhanced VEGFR-3 phosphorylation and subsequent cellular responses as evidenced by the formation of capillary-like structures in PAE cells and proliferation of primary human endothelial cells expressing both receptors. Taken together, these results show for the first time that VEGFR-3 needs to be associated to VEGFR-2 to induce ligand-dependent cellular responses.