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This study aims to uncover the heterogeneity of endothelial cells (ECs) in colorectal cancer (CRC) and their crucial role in angiogenesis, with a special focus on tip cells. Using single-cell RNA sequencing to profile ECs, our data suggests that CRC ECs predominantly exhibit enhanced angiogenesis and decreased antigen presentation, a shift in phenotype largely steered by tip cells. We also observed that an increase in the density and proportion of tip cells correlates with CRC occurrence, progression, and poorer patient prognosis. Furthermore, we identified endothelial cell-specific molecule 1 (ESM1), specifically expressed in tip cells, sustains a VEGFA-KDR-ESM1 positive feedback loop, promoting angiogenesis and CRC proliferation and migration. We also found the enrichment of KDR in tip cells and spotlight a unique long-tail effect in VEGFA expression: while VEGFA is primarily expressed by epithelial cells, the highest level of VEGFA expression is found in individual myeloid cells. Moreover, we observed that effective PD-1 blockade immunotherapy significantly reduced tip cells, disrupting the VEGFA-KDR-ESM1 positive feedback loop in the process. Our investigation into the heterogeneity of ECs in CRC at a single-cell level offers important insights that may contribute to the development of more effective immunotherapies targeting tip cells in CRC.
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Neoplasias Colorrectales , Células Endoteliales , Neovascularización Patológica , Factor A de Crecimiento Endotelial Vascular , Animales , Humanos , Ratones , Angiogénesis/metabolismo , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Neoplasias Colorrectales/patología , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/genética , Células Endoteliales/metabolismo , Células Endoteliales/patología , Regulación Neoplásica de la Expresión Génica , Neovascularización Patológica/metabolismo , Neovascularización Patológica/patología , Neovascularización Patológica/genética , Análisis de la Célula Individual , Factor A de Crecimiento Endotelial Vascular/metabolismo , Factor A de Crecimiento Endotelial Vascular/genética , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genéticaRESUMEN
Angiogenesis is a complex, highly-coordinated and multi-step process of new blood vessel formation from pre-existing blood vessels. When initiated, the sprouting process is spearheaded by the specialized endothelial cells (ECs) known as tip cells, which guide the organization of accompanying stalk cells and determine the function and morphology of the finally-formed blood vessels. Recent studies indicate that the orchestration and coordination of angiogenesis involve dynamic tip cell selection, which is the competitive selection of cells to lead the angiogenic sprouts. Therefore, this review attempt to summarize the underlying mechanisms involved in tip cell specification in a dynamic manner to enable readers to gain a systemic and overall understanding of tip cell formation, involving cooperative interaction of cell rearrangement with Notch and YAP/TAZ signaling. Various mechanical and chemical signaling cues are integrated to ensure the right number of cells at the right place during angiogenesis, thereby precisely orchestrating morphogenic functions that ensure correct patterning of blood vessels. Video Abstract.
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Células Endoteliales , Transducción de Señal , MorfogénesisRESUMEN
Vascularization plays a significant role in promoting the expedited process of bone regeneration while also enhancing the stability and viability of artificial bone implants. Although titanium alloy scaffolds were designed to mimic the porous structure of human bone tissues to facilitate vascularization in bone repair, their biological inertness restricted their broader utilization. The unique attribute of Metal-organic framework (MOF) MIL-53(Fe), known as "breathing", can facilitate the efficient adsorption of extracellular matrix proteins and thus provide the possibility for efficient interaction between scaffolds and cell adhesion molecules, which helps improve the bioactivity of the titanium alloy scaffolds. In this study, MIL-53(Fe) was synthesized in situ on the scaffold after hydrothermal treatment. The MIL-53(Fe) endowed the scaffold with superior protein absorption ability and preferable biocompatibility. The scaffolds have been shown to possess favorable osteogenesis and angiogenesis inducibility. It was indicated that MIL-53(Fe) modulated the mechanotransduction process of endothelial cells and induced increased cell stiffness by promoting the adsorption of adhesion-mediating extracellular matrix proteins to the scaffold, such as laminin, fibronectin, and perlecan et al., which contributed to the activation of the endothelial tip cell phenotype at sprouting angiogenesis. Therefore, this study effectively leveraged the intrinsic "breathing" properties of MIL-53 (Fe) to enhance the interaction between titanium alloy scaffolds and vascular endothelial cells, thereby facilitating the vascularization inducibility of the scaffold, particularly during the sprouting angiogenesis phase. This study indicates that MIL-53(Fe) coating represents a promising strategy to facilitate accelerated and sufficient vascularization and uncovers the scaffold-vessel interaction from a biomechanical perspective.
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Neovascularización Fisiológica , Andamios del Tejido , Titanio , Titanio/química , Humanos , Andamios del Tejido/química , Neovascularización Fisiológica/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Estructuras Metalorgánicas/química , Estructuras Metalorgánicas/farmacología , Materiales Biocompatibles/química , Materiales Biocompatibles/farmacología , Osteogénesis/efectos de los fármacos , Aleaciones/química , Células Endoteliales de la Vena Umbilical Humana , Prótesis e Implantes , Mecanotransducción Celular , Adhesión Celular/efectos de los fármacos , Ingeniería de Tejidos/métodosRESUMEN
Postembryonic angiogenesis is mainly induced by various proangiogenic factors derived from the original vascular network. Previous studies have shown that the role of Ang-2 in angiogenesis is controversial. Tip cells play a vanguard role in angiogenesis and exhibit a transdifferentiated phenotype under the action of angiogenic factors. However, whether Ang-2 promotes the transformation of endothelial cells to tip cells remains unknown. Our study found that miR-221-3p was highly expressed in HCMECs cultured for 4 h under hypoxic conditions (1% O2 ). Moreover, miR-221-3p overexpression inhibited HCMECs proliferation and tube formation, which may play an important role in hypoxia-induced angiogenesis. By target gene prediction, we further demonstrated that Ang-2 was a downstream target of miR-221-3p and miR-221-3p overexpression inhibited Ang-2 expression in HCMECs under hypoxic conditions. Subsequently, qRT-PCR and western blotting methods were performed to analyse the role of miR-221-3p and Ang-2 on the regulation of tip cell marker genes. MiR-221-3p overexpression inhibited CD34, IGF1R, IGF-2 and VEGFR2 proteins expression while Ang-2 overexpression induced CD34, IGF1R, IGF-2 and VEGFR2 expression in HCMECs under hypoxic conditions. In addition, we further confirmed that Ang-2 played a dominant role in miR-221-3p inhibitors promoting the transformation of HCMECs to tip cells by using Ang-2 shRNA to interfere with miR-221-3p inhibitor-treated HCMECs under hypoxic conditions. Finally, we found that miR-221-3p expression was significantly elevated in both serum and myocardial tissue of AMI rats. Hence, our data showed that miR-221-3p may inhibit angiogenesis after acute myocardial infarction by targeting Ang-2 to inhibit the transformation of HCMECs to tip cells.
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MicroARNs , Animales , Ratas , Células Endoteliales/metabolismo , Hipoxia/metabolismo , Factor II del Crecimiento Similar a la Insulina/metabolismo , MicroARNs/metabolismo , HumanosRESUMEN
Angiogenesis plays an essential role in embryonic development, organ remodeling, wound healing, and is also associated with many human diseases. The process of angiogenesis in the brain during development is well characterized in animal models, but little is known about the process in the mature brain. Here, we use a tissue-engineered post-capillary venule (PCV) model incorporating stem cell derived induced brain microvascular endothelial-like cells (iBMECs) and pericyte-like cells (iPCs) to visualize the dynamics of angiogenesis. We compare angiogenesis under two conditions: in response to perfusion of growth factors and in the presence of an external concentration gradient. We show that both iBMECs and iPCs can serve as tip cells leading angiogenic sprouts. More importantly, the growth rate for iPC-led sprouts is about twofold higher than for iBMEC-led sprouts. Under a concentration gradient, angiogenic sprouts show a small directional bias toward the high growth factor concentration. Overall, pericytes exhibited a broad range of behavior, including maintaining quiescence, co-migrating with endothelial cells in sprouts, or leading sprout growth as tip cells.
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Células Endoteliales , Neovascularización Fisiológica , Animales , Humanos , Vénulas , Células Endoteliales/metabolismo , Neovascularización Fisiológica/fisiología , Encéfalo , CapilaresRESUMEN
BACKGROUND: Ocular neovascularization is a leading cause of blindness and visual impairment. While intravitreal anti-VEGF agents can be effective, they do have several drawbacks, such as endophthalmitis and drug resistance. Additional studies are necessary to explore alternative therapeutic targets. METHODS: Bioinformatics analysis and quantitative RT-PCR were used to detect and verify the FSCN1 expression levels in oxygen-induced retinopathy (OIR) and laser-induced choroidal neovascularization (CNV) mice model. Transwell, wound scratching, tube formation, three-dimensional bead sprouting assay, rhodamine-phalloidin staining, Isolectin B4 staining and immunofluorescent staining were conducted to detect the role of FSCN1 and its oral inhibitor NP-G2-044 in vivo and vitro. HPLC-MS/MS analysis, cell apoptosis assay, MTT assay, H&E and tunnel staining, visual electrophysiology testing, visual cliff test and light/dark transition test were conducted to assess the pharmacokinetic and security of NP-G2-044 in vivo and vitro. Co-Immunoprecipitation, qRT-PCR and western blot were conducted to reveal the mechanism of FSCN1 and NP-G2-044 mediated pathological ocular neovascularization. RESULTS: We discovered that Fascin homologue 1 (FSCN1) is vital for angiogenesis both in vitro and in vivo, and that it is highly expressed in oxygen-induced retinopathy (OIR) and laser-induced choroidal neovascularization (CNV). We found that NP-G2-044, a small-molecule inhibitor of FSCN1 with oral activity, can impede the sprouting, migration, and filopodia formation of cultured endothelial cells. Oral NP-G2-044 can effectively and safely curb the development of OIR and CNV, and increase efficacy while overcoming anti-VEGF resistance in combination with intravitreal aflibercept (Eylea) injection. CONCLUSION: Collectively, FSCN1 inhibition could serve as a promising therapeutic approach to block ocular neovascularization.
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Neovascularización Coroidal , Enfermedades de la Retina , Animales , Ratones , Apoptosis , Neovascularización Coroidal/tratamiento farmacológico , Células Endoteliales , Espectrometría de Masas en TándemRESUMEN
BACKGROUND: Pathological neovascularization plays a pivotal role in the onset and progression of tumors and neovascular eye diseases. Despite notable advancements in the development of anti-angiogenic medications that target vascular endothelial growth factor (VEGF) and its receptors (VEGFRs), the occurrence of adverse reactions and drug resistance has somewhat impeded the widespread application of these drugs. Therefore, additional investigations are warranted to explore alternative therapeutic targets. In recent years, owing to the swift advancement of high-throughput sequencing technology, pan-cancer analysis and single-cell sequencing analysis have emerged as pivotal methodologies and focal areas within the domain of omics research, which is of great significance for us to find potential targets related to the regulation of pathological neovascularization. METHODS: Pan-cancer analysis and scRNA-seq data analysis were employed to forecast the association between Actin filament-associated protein 1 like 1 (AFAP1L1) and the development of tumors and endothelial cells. Tumor xenograft model and ocular pathological neovascularization model were constructed as well as Isolectin B4 (IsoB4) staining and immunofluorescence staining were used to assess the effects of AFAP1L1 on the progression of neoplasms and neovascular eye diseases in vivo. Transwell assay, wound scratch assay, tube forming assay, three-dimensional germination assay, and rhodamine-phalloidin staining were used to evaluate the impact of AFAP1L1 on human umbilical vein endothelial cells (HUVECs) function in vitro; Dual luciferase reporting, qRT-PCR and western blot were used to investigate the upstream and downstream mechanisms of pathological neovascularization mediated by AFAP1L1. RESULTS: Our investigation revealed that AFAP1L1 plays a crucial role in promoting the development of various tumors and demonstrates a strong correlation with endothelial cells. Targeted suppression of AFAP1L1 specifically in endothelial cells in vivo proves effective in inhibiting tumor formation and ocular pathological neovascularization. Mechanistically, AFAP1L1 functions as a hypoxia-related regulatory protein that can be activated by HIF-1α. In vitro experiments demonstrated that reducing AFAP1L1 levels can reverse hypoxia-induced excessive angiogenic capacity in HUVECs. The principal mechanism of angiogenesis inhibition entails the regulation of tip cell behavior through the YAP-DLL4-NOTCH axis. CONCLUSION: In conclusion, AFAP1L1, a newly identified hypoxia-related regulatory protein, can be activated by HIF-1α. Inhibiting AFAP1L1 results in the inhibition of angiogenesis by suppressing the germination of endothelial tip cells through the YAP-DLL4-NOTCH axis. This presents a promising therapeutic target to halt the progression of tumors and neovascular eye disease.
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Proteínas Adaptadoras Transductoras de Señales , Células Endoteliales , Neovascularización Patológica , Humanos , Inhibidores de la Angiogénesis , Proteínas de Unión al Calcio , Transducción de Señal , Factor A de Crecimiento Endotelial Vascular , AnimalesRESUMEN
Endothelial tip cells are essential for VEGF-induced angiogenesis, but underlying mechanisms are elusive. The Ena/VASP protein family, consisting of EVL, VASP, and Mena, plays a pivotal role in axon guidance. Given that axonal growth cones and endothelial tip cells share many common features, from the morphological to the molecular level, we investigated the role of Ena/VASP proteins in angiogenesis. EVL and VASP, but not Mena, are expressed in endothelial cells of the postnatal mouse retina. Global deletion of EVL (but not VASP) compromises the radial sprouting of the vascular plexus in mice. Similarly, endothelial-specific EVL deletion compromises the radial sprouting of the vascular plexus and reduces the endothelial tip cell density and filopodia formation. Gene sets involved in blood vessel development and angiogenesis are down-regulated in EVL-deficient P5-retinal endothelial cells. Consistently, EVL deletion impairs VEGF-induced endothelial cell proliferation and sprouting, and reduces the internalization and phosphorylation of VEGF receptor 2 and its downstream signaling via the MAPK/ERK pathway. Together, we show that endothelial EVL regulates sprouting angiogenesis via VEGF receptor-2 internalization and signaling.
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Moléculas de Adhesión Celular/fisiología , Células Endoteliales , Neovascularización Fisiológica , Receptor 2 de Factores de Crecimiento Endotelial Vascular , Animales , Células Endoteliales/metabolismo , Ratones , Morfogénesis , Transducción de Señal , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genética , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Endothelial tip cell specialization plays an essential role in angiogenesis, which is tightly regulated by the complicated gene regulatory network. Circular RNA (circRNA) is a type of covalently closed non-coding RNA that regulates gene expression in eukaryotes. Here, we report that the levels of circMET expression are significantly upregulated in the retinas of mice with oxygen-induced retinopathy, choroidal neovascularization, and diabetic retinopathy. circMET silencing significantly reduces pathological angiogenesis and inhibits tip cell specialization in vivo. circMET silencing also decreases endothelial migration and sprouting in vitro. Mechanistically, circMET regulates endothelial sprouting and pathological angiogenesis by acting as a scaffold to enhance the interaction between IGF2BP2 and NRARP/ESM1. Clinically, circMET is significantly upregulated in the clinical samples of the patients of diabetic retinopathy. circMET silencing could reduce diabetic vitreous-induced endothelial sprouting and retinal angiogenesis in vivo. Collectively, these data identify a circRNA-mediated mechanism that coordinates tip cell specialization and pathological angiogenesis. circMET silencing is an exploitable therapeutic approach for the treatment of neovascular diseases.
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Neovascularización Coroidal , Retinopatía Diabética , Animales , Neovascularización Coroidal/genética , Retinopatía Diabética/tratamiento farmacológico , Retinopatía Diabética/genética , Células Endoteliales/metabolismo , Humanos , Ratones , Ratones Endogámicos C57BL , ARN Circular/genética , Proteínas de Unión al ARN/metabolismo , Retina/metabolismoRESUMEN
Although somatic cells play an integral role in animal gametogenesis, their organization and function are usually poorly characterized, especially in non-model systems. One such example is a peculiar cell found in leech ovaries - the apical cell (AC). A single AC can be found at the apical tip of each ovary cord, the functional unit of leech ovaries, where it is surrounded by other somatic and germline cells. The AC is easily distinguished due to its enormous size and its numerous long cytoplasmic projections that penetrate the space between neighboring cells. It is also characterized by a prominent accumulation of mitochondria, Golgi complexes and electron-dense vesicles. ACs are also enriched in cytoskeleton, mainly in form of intermediate filaments. Additionally, the AC is connected to neighboring cells via junctions that structurally resemble hemidesmosomes. In spite of numerous descriptive data about the AC, its functions remain poorly understood. Its suggested functions include a role in forming skeleton for the germline cells, and a role in defining a niche for germline stem cells. The latter is more speculative, since germline stem cells have not been identified in leech ovaries. Somatic cells with similar morphological properties to those of the AC have been found in gonads of nematodes - the distal tip cell - and in insects - Verson's cell, hub cells and cap cells. In the present article we summarize information about the AC structure and its putative functions. AC is compared with other well-described somatic cells with potentially similar roles in gametogenesis.
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Sanguijuelas/citología , Ovario/citología , Animales , Núcleo Celular/ultraestructura , Citoplasma/ultraestructura , Femenino , Oogénesis , Ovario/fisiología , Ovario/ultraestructura , Nicho de Células MadreRESUMEN
BACKGROUND: Crosstalk between neoplastic and stromal cells fosters prostate cancer (PCa) progression and dissemination. Insight in cell-to-cell communication networks provides new therapeutic avenues to mold processes that contribute to PCa tumor microenvironment (TME) alterations. Here we performed a detailed characterization of PCa tumor endothelial cells (TEC) to delineate intercellular crosstalk between TEC and the PCa TME. METHODS: TEC isolated from 67 fresh radical prostatectomy (RP) specimens underwent multi-omic ex vivo characterization as well as orthogonal validation of both TEC functions and key markers by immunohistochemistry (IHC) and immunofluorescence (IF). To identify cell-cell interaction targets in TEC, we performed single-cell RNA sequencing (scRNA-seq) in four PCa patients who underwent a RP to catalogue cellular TME composition. Targets were cross-validated using IHC, publicly available datasets, cell culture expriments as well as a PCa xenograft mouse model. RESULTS: Compared to adjacent normal endothelial cells (NEC) bulk RNA-seq analysis revealed upregulation of genes associated with tumor vasculature, collagen modification and extracellular matrix remodeling in TEC. PTGIR, PLAC9, CXCL12 and VDR were identified as TEC markers and confirmed by IF and IHC in an independent patient cohort. By scRNA-seq we identified 27 cell (sub)types, including endothelial cells (EC) with arterial, venous and immature signatures, as well as angiogenic tip EC. A focused molecular analysis revealed that arterial TEC displayed highest CXCL12 mRNA expression levels when compared to all other TME cell (sub)populations and showed a negative prognostic role. Receptor-ligand interaction analysis predicted interactions between arterial TEC derived CXCL12 and its cognate receptor CXCR4 on angiogenic tip EC. CXCL12 was in vitro and in vivo validated as actionable TEC target by highlighting the vessel number- and density- reducing activity of the CXCR4-inhibitor AMD3100 in murine PCa as well as by inhibition of TEC proliferation and migration in vitro. CONCLUSIONS: Overall, our comprehensive analysis identified novel PCa TEC targets and highlights CXCR4/CXCL12 interaction as a potential novel target to interfere with tumor angiogenesis in PCa.
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Próstata , Neoplasias de la Próstata , Animales , Línea Celular Tumoral , Proliferación Celular , Quimiocina CXCL12/genética , Quimiocina CXCL12/metabolismo , Células Endoteliales/metabolismo , Humanos , Masculino , Ratones , Próstata/metabolismo , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Receptores CXCR4/genética , Receptores CXCR4/metabolismo , Receptores de Epoprostenol , Microambiente TumoralRESUMEN
Sprouting angiogenesis is a highly coordinately process controlled by vascular endothelial growth factor receptor (VEGFR)-Notch signaling. Here we investigated whether Tripartite motif-containing 28 (TRIM28), which is an epigenetic modifier implicated in gene transcription and cell differentiation, is essential to mediate sprouting angiogenesis. We observed that knockdown of TRIM28 ortholog in zebrafish resulted in developmental vascular defect with disorganized and reduced vasculatures. Consistently, TRIM28 knockdown inhibited angiogenic sprouting of cultured endothelial cells (ECs), which exhibited increased mRNA levels of VEGFR1, Delta-like (DLL) 3, and Notch2 but reduced levels of VEGFR2, DLL1, DLL4, Notch1, Notch3, and Notch4.The regulative effects of TRIM28 on these angiogenic factors were partially mediated by hypoxia-inducible factor 1 α (HIF-1α) and recombination signal-binding protein for immunoglobulin kappa J region (RBPJκ). In vitro DNA-binding assay showed that TRIM28 knockdown increased the association of RBPJκ with DNA sequences containing HIF-1α-binding sites. Moreover, the phosphorylation of TRIM28 was controlled by VEGF and Notch1 through a mechanism involving RBPJκ-dual-specificity phosphatase (DUSP)-p38 MAPK, indicating a negative feedback mechanism. These findings established TRIM28 as a crucial regulator of VEGFR-Notch signaling circuit through HIF-1α and RBPJκ in EC sprouting angiogenesis.
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Neovascularización Fisiológica , Transducción de Señal , Proteína 28 que Contiene Motivos Tripartito/metabolismo , Animales , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Factor 1 Inducible por Hipoxia/metabolismo , Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Masculino , Ratas , Ratas Sprague-Dawley , Receptores Notch/genética , Receptores Notch/metabolismo , Receptores de Factores de Crecimiento Endotelial Vascular/genética , Receptores de Factores de Crecimiento Endotelial Vascular/metabolismo , Proteína 28 que Contiene Motivos Tripartito/genética , Pez Cebra , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Proliferative retinopathies, such as diabetic retinopathy and retinopathy of prematurity, are leading causes of vision impairment. A common feature is a loss of retinal capillary vessels resulting in hypoxia and neuronal damage. The oxygen-induced retinopathy model is widely used to study revascularization of an ischemic area in the mouse retina. The presence of endothelial tip cells indicates vascular recovery; however, their quantification relies on manual counting in microscopy images of retinal flat mount preparations. Recent advances in deep neural networks (DNNs) allow the automation of such tasks. We demonstrate a workflow for detection of tip cells in retinal images using the DNN-based Single Shot Detector (SSD). The SSD was designed for detection of objects in natural images. We adapt the SSD architecture and training procedure to the tip cell detection task and retrain the DNN using labeled tip cells in images of fluorescently stained retina flat mounts. Transferring knowledge from the pretrained DNN and extensive data augmentation reduced the amount of required labeled data. Our system shows a performance comparable to the human level, while providing highly consistent results. Therefore, such a system can automate counting of tip cells, a readout frequently used in retinopathy research, thereby reducing routine work for biomedical experts.
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Aprendizaje Profundo , Enfermedades de la Retina , Animales , Humanos , Ratones , Redes Neurales de la Computación , Oxígeno , Enfermedades de la Retina/inducido químicamente , Vasos RetinianosRESUMEN
Purinergic P2 receptors are critical regulators of several functions within the vascular system, including platelet aggregation, vascular inflammation, and vascular tone. However, a role for ATP release and P2Y receptor signalling in angiogenesis remains poorly defined. Here, we demonstrate that blood vessel growth is controlled by P2Y2 receptors. Endothelial sprouting and vascular tube formation were significantly dependent on P2Y2 expression and inhibition of P2Y2 using a selective antagonist blocked microvascular network generation. Mechanistically, overexpression of P2Y2 in endothelial cells induced the expression of the proangiogenic molecules CXCR4, CD34, and angiopoietin-2, while expression of VEGFR-2 was decreased. Interestingly, elevated P2Y2 expression caused constitutive phosphorylation of ERK1/2 and VEGFR-2. However, stimulation of cells with the P2Y2 agonist UTP did not influence sprouting unless P2Y2 was constitutively expressed. Finally, inhibition of VEGFR-2 impaired spontaneous vascular network formation induced by P2Y2 overexpression. Our data suggest that P2Y2 receptors have an essential function in angiogenesis, and that P2Y2 receptors present a therapeutic target to regulate blood vessel growth.
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Células Endoteliales/metabolismo , Endotelio Vascular/crecimiento & desarrollo , Neovascularización Fisiológica/fisiología , Receptores Purinérgicos P2Y2/metabolismo , Angiopoyetina 2/biosíntesis , Antígenos CD34/biosíntesis , Células Cultivadas , Humanos , Proteína Quinasa 1 Activada por Mitógenos/biosíntesis , Proteína Quinasa 3 Activada por Mitógenos/biosíntesis , Fosforilación/fisiología , Agregación Plaquetaria/fisiología , Antagonistas del Receptor Purinérgico P2Y/farmacología , Interferencia de ARN , ARN Interferente Pequeño/genética , Receptores CXCR4/biosíntesis , Receptores Purinérgicos P2Y2/genética , Receptor 2 de Factores de Crecimiento Endotelial Vascular/biosíntesisRESUMEN
Blood vessels are essential for the formation and maintenance of almost all functional tissues. They play fundamental roles in the supply of oxygen and nutrition, as well as development and morphogenesis. Vascular endothelial cells are the main factor in blood vessel formation. Recently, research findings showed heterogeneity in vascular endothelial cells in different tissue/organs. Endothelial cells alter their gene expressions depending on their cell fate or angiogenic states of vascular development in normal and pathological processes. Studies on gene regulation in endothelial cells demonstrated that the activator protein 1 (AP-1) transcription factors are implicated in angiogenesis and vascular development. In particular, it has been revealed that JunB (a member of the AP-1 transcription factor family) is transiently induced in endothelial cells at the angiogenic frontier and controls them on tip cells specification during vascular development. Moreover, JunB plays a role in tissue-specific vascular maturation processes during neurovascular interaction in mouse embryonic skin and retina vasculatures. Thus, JunB appears to be a new angiogenic factor that induces endothelial cell migration and sprouting particularly in neurovascular interaction during vascular development. In this review, we discuss the recently identified role of JunB in endothelial cells and blood vessel formation.
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Inductores de la Angiogénesis/metabolismo , Movimiento Celular , Células Endoteliales/metabolismo , Neovascularización Fisiológica , Factor de Transcripción AP-1/metabolismo , Factores de Transcripción/metabolismo , Animales , HumanosRESUMEN
Angiogenesis is largely driven by motile endothelial tip-cells capable of invading avascular tissue domains and enabling new vessel formation. Highly responsive to Vascular Endothelial Growth-Factor-A (VEGFA), endothelial tip-cells also suppress angiogenic sprouting in adjacent stalk cells, and thus have been a primary therapeutic focus in addressing neovascular pathologies. Surprisingly, however, there remains a paucity of specific endothelial tip-cell markers. Here, we employ transcriptional profiling and a lacZ reporter allele to identify Kcne3 as an early and selective endothelial tip-cell marker in multiple angiogenic contexts. In development, Kcne3 expression initiates during early phases of angiogenesis (E9) and remains specific to endothelial tip-cells, often adjacent to regions expressing VEGFA. Consistently, Kcne3 activation is highly responsive to exogenous VEGFA but maintains tip-cell specificity throughout normal retinal angiogenesis. We also demonstrate endothelial tip-cell selectivity of Kcne3 in several injury and tumor models. Together, our data show that Kcne3 is a unique marker of sprouting angiogenic tip-cells and offers new opportunities for investigating and targeting this cell type.
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Células Endoteliales/fisiología , Neovascularización Patológica/genética , Neovascularización Fisiológica/genética , Canales de Potasio con Entrada de Voltaje/genética , Factor A de Crecimiento Endotelial Vascular/fisiología , Animales , Animales Recién Nacidos , Diferenciación Celular/genética , Células Cultivadas , Retinopatía Diabética/genética , Retinopatía Diabética/patología , Embrión de Mamíferos , Células Endoteliales/patología , Femenino , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Transgénicos , Morfogénesis/genética , Neovascularización Patológica/metabolismo , Embarazo , Retina/metabolismo , Retina/patología , Vasos Retinianos/metabolismo , Vasos Retinianos/patologíaRESUMEN
A drug undergoes several in silico, in vitro, ex vivo and in vivo assays before entering into the clinical trials. In 2014, it was reported that only 32% of drugs are likely to make it to Phase-3 trials, and overall, only one in 10 drugs makes it to the market. Therefore, enhancing the precision of pre-clinical trial models could reduce the number of failed clinical trials and eventually time and financial burden in health sciences. In order to attempt the above, in the present study, we have shown that aortic ex-plants isolated from different stages of chick embryo and different regions of the aorta (pulmonary and systemic) have differential sprouting potential and response to angiogenesis modulatory drugs. Aorta isolated from HH37 staged chick embryo showed 16% (pâ¯<â¯0.001) and 11% (pâ¯<â¯0.001) increase in the number of tip cells at 72â¯h of culture compared to that of HH35 and HH29 respectively. The ascending order of the number of tip cells was found as central (Gen II), proximal (Gen I) and distal (Gen III) in a virtual zonal segmentation of endothelial sprouting. The HH37 staged aortas displayed differential responses to pro- and anti-angiogenic drugs like Vascular endothelial growth factor (VEGF), nitric oxide donor (spNO), and bevacizumab (avastin), thalidomide respectively. The human placenta tissue-culture however evinced endothelial sprouting only on day 12, with a gradual decrease in the number of tip cells until 21â¯days. In summary, this study provides an avant-garde angiogenic model emphasized on tip cells that would enhance the precision to test next-generation angiogenic drugs.
Asunto(s)
Inductores de la Angiogénesis/farmacología , Inhibidores de la Angiogénesis/farmacología , Aorta Torácica/embriología , Bioensayo , Células Endoteliales/efectos de los fármacos , Neovascularización Fisiológica/efectos de los fármacos , Placenta/irrigación sanguínea , Animales , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Embrión de Pollo , Células Endoteliales/fisiología , Femenino , Humanos , Embarazo , Reproducibilidad de los Resultados , Factores de Tiempo , Técnicas de Cultivo de TejidosRESUMEN
Cell migration is a fundamental biological process involved in for example embryonic development, immune system and wound healing. Cell migration is also a key step in cancer metastasis and the human copper chaperone Atox1 was recently found to facilitate this process in breast cancer cells. To explore the role of the copper chaperone in other cell migration processes, we here investigated the putative involvement of an Atox1 homolog in Caenorhabditis elegans, CUC-1, in distal tip cell migration, which is a key process during the development of the C. elegans gonad. Using knock-out worms, in which the cuc-1 gene was removed by CRISPR-Cas9 technology, we probed life span, brood size, as well as distal tip cell migration in the absence or presence of supplemented copper. Upon scoring of gonads, we found that cuc-1 knock-out, but not wild-type, worms exhibited distal tip cell migration defects in approximately 10-15% of animals and, had a significantly reduced brood size. Importantly, the distal tip cell migration defect was rescued by a wild-type cuc-1 transgene provided to cuc-1 knock-out worms. The results obtained here for C. elegans CUC-1 imply that Atox1 homologs, in addition to their well-known cytoplasmic copper transport, may contribute to developmental cell migration processes.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Chaperonas Moleculares/metabolismo , Animales , Caenorhabditis elegans/citología , Caenorhabditis elegans/crecimiento & desarrollo , Proteínas de Caenorhabditis elegans/genética , Movimiento Celular , Cobre/metabolismo , Proteínas Transportadoras de Cobre/genética , Proteínas Transportadoras de Cobre/metabolismo , Humanos , Chaperonas Moleculares/genéticaRESUMEN
Extrinsic branching factors promote the elongation and migration of tubular organs. In the Drosophila tracheal system, Branchless (Drosophila FGF) stimulates the branching program by specifying tip cells that acquire motility and lead branch migration to a specific destination. Tip cells have two alternative cell fates: the terminal cell (TC), which produces long cytoplasmic extensions with intracellular lumen, and the fusion cell (FC), which mediates branch connections to form tubular networks. How Branchless controls this specification of cells with distinct shapes and behaviors is unknown. Here we report that this cell type diversification involves the modulation of FGF signaling by the zinc-finger protein Escargot (Esg), which is expressed in the FC and is essential for its specification. The dorsal branch begins elongation with a pair of tip cells with high FGF signaling. When the branch tip reaches its final destination, one of the tip cells becomes an FC and expresses Esg. FCs and TCs differ in their response to FGF: TCs are attracted by FGF, whereas FCs are repelled. Esg suppresses ERK signaling in FCs to control this differential migratory behavior.
Asunto(s)
Diferenciación Celular/genética , Linaje de la Célula/genética , Proteínas de Drosophila/fisiología , Drosophila melanogaster , Factores de Crecimiento de Fibroblastos/metabolismo , Morfogénesis/genética , Tráquea/embriología , Animales , Fusión Celular , Movimiento Celular/genética , Drosophila melanogaster/embriología , Drosophila melanogaster/genética , Embrión no Mamífero , Factores de Crecimiento de Fibroblastos/antagonistas & inhibidores , Factores de Crecimiento de Fibroblastos/genética , Regulación del Desarrollo de la Expresión Génica , Sistema de Señalización de MAP Quinasas/genética , Transducción de Señal/genética , Tráquea/citología , Tráquea/metabolismoRESUMEN
Many stem cell niches contain support cells that increase contact with stem cells by enwrapping them in cellular processes. One example is the germ stem cell niche in C. elegans, which is composed of a single niche cell termed the distal tip cell (DTC) that extends cellular processes, constructing an elaborate plexus that enwraps germ stem cells. To identify genes required for plexus formation and to explore the function of this specialized enwrapping behavior, a series of targeted and tissue-specific RNAi screens were performed. Here we identify genes that promote stem cell enwrapment by the DTC plexus, including a set that specifically functions within the DTC, such as the chromatin modifier lin-40/MTA1, and others that act within the germline, such as the 14-3-3 signaling protein par-5. Analysis of genes that function within the germline to mediate plexus development reveal that they are required for expansion of the germ progenitor zone, supporting the emerging idea that germ stem cells signal to the niche to stimulate enwrapping behavior. Examination of wild-type animals with asymmetric plexus formation and animals with reduced DTC plexus elaboration via loss of two candidates including lin-40 indicate that cellular enwrapment promotes GLP-1/Notch signaling and germ stem cell fate. Together, our work identifies novel regulators of cellular enwrapment and suggests that reciprocal signaling between the DTC niche and the germ stem cells promotes enwrapment behavior and stem cell fate.