RESUMEN
Poly(ethylene glycol)-b-polyphosphoester (PEG-b-PPE) block copolymer nanoparticles are promising carriers for poorly water soluble drugs. To enhance the drug loading capacity and efficiency of such micelles, a strategy was investigated for increasing the lipophilicity of the PPE block of these PEG-b-PPE amphiphilic copolymers. A PEG-b-PPE copolymer bearing pendant vinyl groups along the PPE block was synthesized and then modified by thiol-ene click reaction with thiols bearing either a long linear alkyl chain (dodecyl) or a tocopherol moiety. Ketoconazole was used as model for hydrophobic drugs. Comparison of the drug loading with PEG-b-PPE bearing shorter pendant groups is reported evidencing the key role of the structure of the pendant group on the PPE backbone. Finally, a first evidence of the biocompatibility of these novel PEG-b-PPE copolymers was achieved by performing cytotoxicity tests. The PEG-b-PPE derived by tocopherol was evidenced as particularly promising as delivery system of poorly water-soluble drugs.
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Portadores de Fármacos , Diseño de Fármacos , Micelas , Poliésteres , Polietilenglicoles , Portadores de Fármacos/química , Portadores de Fármacos/uso terapéutico , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Cetoconazol/química , Cetoconazol/uso terapéutico , Poliésteres/química , Poliésteres/uso terapéutico , Polietilenglicoles/química , Polietilenglicoles/uso terapéuticoRESUMEN
Functional synthetic polymers are frequently explored for their use in the biomedical field. To fulfill the stringent demands of biodegradability and compatibility, the materials need to be versatile and tunable. Post-modification is often considered challenging for well-known degradable materials like poly(lactic acid) because of their chemical inertness. In this work a procedure is proposed to produce densely functionalized polymer particles using oligomeric precursors synthesized via the Morita-Baylis-Hillman reaction. This allows for a variety of post-modification reactions to serve bio-conjugation or tuning of the material properties. The particles are subjected to basic media and found to be degradable. Furthermore, cytotoxicity tests confirm good biocompatibility. Finally, as a proof of concept to demonstrate the versatility of the particles, post-modification reactions are carried out through the formation of imines.
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Polímeros/síntesis química , Animales , Materiales Biocompatibles/síntesis química , Materiales Biocompatibles/química , Materiales Biocompatibles/farmacología , Línea Celular , Supervivencia Celular/efectos de los fármacos , Fibroblastos/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Humanos , Estructura Molecular , Tamaño de la Partícula , Polimerizacion , Polímeros/química , Polímeros/farmacología , Propiedades de Superficie , PorcinosRESUMEN
A major conceptual breakthrough in cell signaling has been the finding of EV as new biomarker shuttles in body fluids. Now, one of the major challenges in using these nanometer-sized biological entities as diagnostic marker is the development of translational methodologies to profile them. SPR offers a promising label-free and real time platform with a high potential for biomarker detection. Therefore, we aimed to develop a uniform SPR methodology to detect specific surface markers on EV derived from patient with CHD. EVs having an approximate size range between 30 and 100 nm (~48.5%) and 100-300 nm (~51.5%) were successfully isolated. The biomarker profile of EV was verified using immunogold labeling, ELISA and SPR. Using SPR, we demonstrated an increased binding of EV derived from patients with CHD to anti-ICAM-1 antibodies as compared to EV from healthy donors. Our current findings open up novel opportunities for in-depth and label-free investigation of EV.
Asunto(s)
Biomarcadores , Células Endoteliales , Vesículas Extracelulares , Resonancia por Plasmón de Superficie , Enfermedad Coronaria , Humanos , Inflamación , Nanotecnología/métodosRESUMEN
AIMS: The mechanisms of monocyte recruitment to arteriogenic collaterals are largely unknown. We investigated the role of chemokine (C-X-C-motif) ligand 1 (CXCL1) and its cognate receptor, chemokine (C-X-C-motif) receptor 2 (CXCR2) in arteriogenesis. METHODS AND RESULTS: After femoral artery ligation in Sprague-Dawley rats, either native collaterals were harvested or placebo, CXCL1 or CXCR2 blocker was administered via an osmopump. Perfusion recovery was measured with Laser Doppler, leukocyte populations were analyzed by fluorescence-activated cell sorting, and hind limb sections were stained for macrophage marker cluster of differentiation 68 (CD68). In vitro, fluorescent CXCL1 or human acute monocytic leukemia cell line (THP-1) monocytic cells were flown over shear-stressed endothelium. CXCL1 mRNA expression in collaterals was dramatically upregulated already 1 h after ligation (ratio ligated/sham 5.73). CD68 mRNA was upregulated from 12 h until 3 days after ligation (peak ratio ligated/sham 2.65). CXCL1 treatment augmented perfusion recovery at 3 and 7 days (p < 0.05) after ligation, and a significant increase in the number of peri-collateral macrophages was evident concomitantly (p < 0.05). Conversely, CXCR2 antagonist treatment caused a decrease in perfusion recovery both at 7 and 10 days postligation (p = 0.01) and also significantly reduced the number of peri-collateral macrophages (p < 0.05). In vitro, CXCL1 tethered to and was taken up by endothelial cells under shear stress conditions and enhanced THP-1 adherence compared to control (p < 0.05). In contrast, CXCR2 antagonist compromised THP-1 adherence to endothelial cells (p < 0.05). CONCLUSION: CXCL1 presented on the luminal endothelial surface leads to an increase in the number of peri-collateral macrophages, thus improving the arteriogenic response after arterial ligation.
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Arterias/crecimiento & desarrollo , Quimiocina CXCL1/farmacología , Células Musculares/citología , Animales , Células Cultivadas , Quimiocina CXCL1/administración & dosificación , Quimiocina CXCL1/genética , Masculino , ARN Mensajero/genética , Ratas , Ratas Sprague-Dawley , Receptores de Interleucina-8B/antagonistas & inhibidoresRESUMEN
Orthopedic implants such as knee and hip implants are one of the most important types of medical devices. Currently, the surface of the most advanced implants consists of titanium or titanium-alloys with high porosity at the bone-contacting surface leading to superior mechanical properties, excellent biocompatibility, and the capability of inducing osseointegration. However, the increased surface area of porous titanium provides a nidus for bacteria colonization leading to implant-related infections, one of the main reasons for implant failure. Here, two readily applicable titanium-coatings based on hydrophilic carboxybetaine polymers that turn the surface stealth thereby preventing bacterial adhesion and colonization are developed. These coatings are biocompatible, do not affect cell functionality, exhibit great antifouling properties, and do not cause additional inflammation during the healing process. In this way, the coatings can prevent implant-related infections, while at the same time being completely innocuous to its biological environment. Thus, these coating strategies are a promising route to enhance the biocompatibility of orthopedic implants and have a high potential for clinical use, while being easy to implement in the implant manufacturing process.
Asunto(s)
Materiales Biocompatibles Revestidos , Titanio , Titanio/farmacología , Materiales Biocompatibles Revestidos/farmacología , Prótesis e Implantes , Oseointegración , Polímeros , Propiedades de SuperficieRESUMEN
Congenital cardiac abnormalities are, due to their relatively high frequency and severe impact on quality of life, an important focus in cardiovascular research. Recently, various human studies have revealed a high coincidence of VEGF and NOTCH polymorphisms with cardiovascular outflow tract anomalies, such as bicuspid aortic valves and Tetralogy of Fallot, next to predisposition for cardiovascular pathologies, including atherosclerosis and aortic valve calcification. This genetic association between VEGF/NOTCH mutations and congenital cardiovascular defects in humans has been supported by substantial proof from animal models, revealing interaction of both pathways in cellular processes that are crucial for cardiac development. This review focuses on the role of VEGF and NOTCH signaling and their interplay in cardiogenesis with special interest to coronary and outflow tract development. An overview of the association between congenital malformations and VEGF/NOTCH polymorphisms in humans will be discussed along with their potential mechanisms and processes as revealed by transgenic mouse models. The molecular and cellular interaction of VEGF and subsequent Notch-signaling in these processes will be highlighted.
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Diferenciación Celular , Cardiopatías Congénitas/genética , Corazón/embriología , Miocitos Cardíacos/metabolismo , Receptor Notch1/genética , Factor A de Crecimiento Endotelial Vascular/genética , Animales , Aterosclerosis/genética , Vasos Coronarios/embriología , Modelos Animales de Enfermedad , Predisposición Genética a la Enfermedad , Válvulas Cardíacas/embriología , Humanos , Ratones , Mutación , Miocitos Cardíacos/citología , Polimorfismo Genético , Receptor Notch1/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Acute myocardial infarction (AMI) is accompanied by a systemic trauma response that impacts the whole body, including blood. This study addresses whether macrophages, key players in trauma repair, sense and respond to these changes. For this, healthy human monocyte-derived macrophages are exposed to 20% human AMI (n = 50) or control (n = 20) serum and analyzed by transcriptional and multiparameter functional screening followed by network-guided data interpretation and drug repurposing. Results are validated in an independent cohort at functional level (n = 47 AMI, n = 25 control) and in a public dataset. AMI serum exposure results in an overt AMI signature, enriched in debris cleaning, mitosis, and immune pathways. Moreover, gene networks associated with AMI and with poor clinical prognosis in AMI are identified. Network-guided drug screening on the latter unveils prostaglandin E2 (PGE2) signaling as target for clinical intervention in detrimental macrophage imprinting during AMI trauma healing. The results demonstrate pronounced context-induced macrophage reprogramming by the AMI systemic environment, to a degree decisive for patient prognosis. This offers new opportunities for targeted intervention and optimized cardiovascular disease risk management.
Asunto(s)
Macrófagos , Infarto del Miocardio , Humanos , Macrófagos/metabolismo , Infarto del Miocardio/metabolismo , Pronóstico , Redes Reguladoras de GenesRESUMEN
OBJECTIVE: Notch has been implicated in neointima formation as reflected by increased Notch/Jagged expression on vascular injury and the promigratory effect of Notch signaling on smooth muscle cells. Soluble Jagged-1 (sJag1) has been shown to inhibit Notch signaling in vitro; however, its capacity to suppress neointima formation remains unknown. METHODS AND RESULTS: Balloon injury of rat carotid arteries induced Notch1, Notch3, and Jagged-1 expression at days 3 and 14 postinjury. Notch signaling was activated as shown by increased expression of the Notch target gene Herp2. Adenoviral sJag1 (Ad-sJag1) transfection reduced neointima formation in carotid artery and enhanced reendothelialization, whereas adenoviral full-length Jagged-1 (Ad-Fl-Jag1) or LacZ had no effect. Injury-induced Herp2 expression was absent in vessels treated with Ad-sJag1. Consistently, Herp2 expression was reduced in Ad-sJag1-infected or recombinant sJag1 -treated coronary artery smooth muscle cells (CASMCs). Ad-sJag1 had no effect on human umbilical endothelial cell behavior, but it significantly reduced proliferation and migration of CASMCs. Overexpression of Herp2 in sJag1-treated CASMCs rescued the migratory and proliferative capacity in vitro. CONCLUSIONS: Our results demonstrate that sJag1 can inhibit neointima formation after balloon injury by decreasing smooth muscle cell proliferation and migration through interference with Notch-Herp2 signaling.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Proteínas de Unión al Calcio/metabolismo , Arterias Carótidas/metabolismo , Traumatismos de las Arterias Carótidas/prevención & control , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteínas de la Membrana/metabolismo , Receptor Notch1/metabolismo , Receptores Notch/metabolismo , Proteínas Represoras/metabolismo , Transducción de Señal , Túnica Íntima/metabolismo , Análisis de Varianza , Animales , Proteínas de Unión al Calcio/genética , Arterias Carótidas/patología , Traumatismos de las Arterias Carótidas/genética , Traumatismos de las Arterias Carótidas/metabolismo , Traumatismos de las Arterias Carótidas/patología , Movimiento Celular , Proliferación Celular , Modelos Animales de Enfermedad , Células Endoteliales/metabolismo , Células Endoteliales/patología , Hiperplasia , Péptidos y Proteínas de Señalización Intercelular/genética , Proteína Jagged-1 , Proteínas de la Membrana/genética , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patología , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/patología , Ratas , Ratas Sprague-Dawley , Receptor Notch3 , Proteínas Serrate-Jagged , Factores de Tiempo , Transfección , Túnica Íntima/patologíaRESUMEN
The biocompatibility, tunable degradability and broad functionalities of polyphosphoesters and their potential for biomedical applications have stimulated a renewed interest from Chemistry, Medicinal Chemistry and Polymer Sciences. Commercial applications of polyphosphoesters as biomaterials are still hampered because of the time and resource-intensive sourcing of their corresponding monomers, in addition to the corrosive and sensitive nature of their intermediates and by-products. Here, we present a groundbreaking challenge for sourcing the corresponding cyclic phosphate monomers by a different approach. This approach relies on the use of continuous flow technologies to intensify the end-to-end preparation of cyclic phosphate monomers with a semi-continuous modular flow platform. The applied flow technology mitigates both safety and instability issues related to the more classical production of cyclic phosphate monomers. The first flow module allows safe synthesis of a library of cyclic chlorophosphite building blocks and features in-line 31P NMR real-time monitoring. After optimization on the microfluidic scale, this first module is successfully transposed toward mesofluidic scale with a daily throughput of 1.88 kg. Downstream of the first module, a second module is present, allowing the quantitative conversion of cyclic chlorophosphites with molecular oxygen toward chlorophosphate derivatives within seconds. The two modules are concatenable with a downstream semi-batch quench of intermediate chlorophosphate with alcohols, hence affording the corresponding cyclic phosphate monomers. Such a continuous flow setup provides considerable unprecedented advantages to safely and efficiently synthesize a library of versatile high value-added cyclic phosphate monomers at large scale. These freshly produced monomers can be successfully (co)polymerized, using either batch or flow protocols, into well-defined polyphosphoesters with assessed thermal properties and cytotoxicity.
RESUMEN
The DELTA like-4 ligand (DLL4) belongs to the highly conserved NOTCH family and is specifically expressed in the endothelium. DLL4 regulates crucial processes in vascular growth, including endothelial cell (EC) sprouting and arterial specification. Its expression is increased by VEGF-A. In the present study, we show that VEGF-induced DLL4 expression depends on NOTCH activation. VEGF-induced DLL4 expression was prevented by the blockage of NOTCH signaling with γ-secretase or ADAM inhibitors in human cardiac microvascular ECs. Similar to VEGF-A, recombinant DLL4 itself stimulated NOTCH signaling and resulted in up-regulation of DLL4, suggesting a positive feed-forward mechanism. These effects were abrogated by NOTCH inhibitors but not by inhibition of VEGF signaling. NOTCH activation alone suffices to induce DLL4 expression as illustrated by the positive effect of NOTCH intracellular domain (NICD)-1 or -4 overexpression. To discriminate between NICD/RBP-Jκ and FOXC2-regulated DLL4 expression, DLL4 promoter activity was assessed in promoter deletion experiments. NICD induced promoter activity was dependent on RBP-Jκ site but independent of the FOXC2 binding site. Accordingly, constitutively active FOXC2 did not affect DLL4 expression. The notion that the positive feed-forward mechanism might propagate NOTCH activation to neighboring ECs was supported by our observation that DLL4-eGFP-transfected ECs induced DLL4 expression in nontransfected cells in their vicinity. In summary, our data provide evidence for a mechanism by which VEGF or ligand-induced NOTCH signaling up-regulates DLL4 through a positive feed-forward mechanism. By this mechanism, DLL4 could propagate its own expression and enable synchronization of NOTCH expression and signaling between ECs.
Asunto(s)
Comunicación Celular/fisiología , Vasos Coronarios/metabolismo , Células Endoteliales/metabolismo , Péptidos y Proteínas de Señalización Intercelular/biosíntesis , Receptores Notch/metabolismo , Elementos de Respuesta/fisiología , Transducción de Señal/fisiología , Regulación hacia Arriba/fisiología , Factor A de Crecimiento Endotelial Vascular/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Secretasas de la Proteína Precursora del Amiloide/genética , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Proteínas de Unión al Calcio , Células Cultivadas , Vasos Coronarios/citología , Células Endoteliales/citología , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Humanos , Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas/genética , Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas/metabolismo , Péptidos y Proteínas de Señalización Intercelular/genética , Estructura Terciaria de Proteína , Receptores Notch/genética , Factor A de Crecimiento Endotelial Vascular/genéticaRESUMEN
OBJECTIVE: To elucidate the downstream mechanisms of vascular endothelial growth factor receptor 2 (VEGFR2), a key receptor in angiogenesis, which has been associated with atherosclerotic plaque growth and instability. METHODS AND RESULTS: By using a yeast-2-hybrid assay, we identified A Disintegrin And Metalloprotease 10 (ADAM10) as a novel binding partner of VEGFR2. ADAM10 is a metalloprotease with sheddase activity involved in cell migration; however, its exact function in endothelial cells (ECs), angiogenesis, and atherosclerosis is largely unknown. For the first time to our knowledge, we show ADAM10 expression in human atherosclerotic lesions, associated with plaque progression and neovascularization. We demonstrate ADAM10 expression and activity in ECs to be induced by VEGF; also, ADAM10 mediates the ectodomain shedding of VEGFR2. Furthermore, VEGF induces ADAM10-mediated cleavage of vascular endothelium (VE)-cadherin, which could increase vascular permeability and facilitate EC migration. Indeed, VEGF increases vascular permeability in an ADAM10- and ADAM17-dependent way; inhibition of ADAM10 reduces EC migration and chemotaxis. CONCLUSIONS: These data provide the first evidence of ADAM10 expression in atherosclerosis and neovascularization. ADAM10 plays a functional role in VEGF-induced EC function. These data open perspectives for novel therapeutic interventions in vascular diseases.
Asunto(s)
Proteínas ADAM/fisiología , Secretasas de la Proteína Precursora del Amiloide/fisiología , Aterosclerosis/fisiopatología , Células Endoteliales/fisiología , Proteínas de la Membrana/fisiología , Neovascularización Patológica/fisiopatología , Receptor 2 de Factores de Crecimiento Endotelial Vascular/fisiología , Proteínas ADAM/biosíntesis , Proteína ADAM10 , Secretasas de la Proteína Precursora del Amiloide/biosíntesis , Aterosclerosis/metabolismo , Células Cultivadas , Progresión de la Enfermedad , Humanos , Proteínas de la Membrana/biosíntesis , Neovascularización Patológica/metabolismo , Placa Aterosclerótica/metabolismo , Placa Aterosclerótica/fisiopatologíaRESUMEN
Hemizygous deletion of chromosome 22q11 (del22q11) causes thymic, parathyroid, craniofacial and life-threatening cardiovascular birth defects in 1 in 4,000 infants. The del22q11 syndrome is likely caused by haploinsufficiency of TBX1, but its variable expressivity indicates the involvement of additional modifiers. Here, we report that absence of the Vegf164 isoform caused birth defects in mice, reminiscent of those found in del22q11 patients. The close correlation of birth and vascular defects indicated that vascular dysgenesis may pathogenetically contribute to the birth defects. Vegf interacted with Tbx1, as Tbx1 expression was reduced in Vegf164-deficient embryos and knocked-down vegf levels enhanced the pharyngeal arch artery defects induced by tbx1 knockdown in zebrafish. Moreover, initial evidence suggested that a VEGF promoter haplotype was associated with an increased risk for cardiovascular birth defects in del22q11 individuals. These genetic data in mouse, fish and human indicate that VEGF is a modifier of cardiovascular birth defects in the del22q11 syndrome.
Asunto(s)
Deleción Cromosómica , Síndrome de DiGeorge/genética , Factores de Crecimiento Endotelial/genética , Péptidos y Proteínas de Señalización Intercelular/genética , Linfocinas/genética , Animales , Vasos Sanguíneos/anomalías , Anomalías Congénitas/genética , Cara/anomalías , Ratones , Ratones Noqueados , Neuropilina-1/genética , Isoformas de Proteínas/genética , Cráneo/anomalías , Proteínas de Dominio T Box/genética , Timo/anomalías , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial Vascular , Pez CebraRESUMEN
Substantial research has been devoted to discovering the translational potential of extracellular vesicles (EV) as a reliable liquid biopsy in the diagnosis and monitoring of several life-affecting diseases, including chronic inflammatory diseases (CID). So far, the role of EV in the development of CID remains largely unknown due to the lack of specific tools to separate the disease-associated EV subtypes. Therefore, this study aims to fractionate inflammation-associated EV (sub)populations using a two-step separation strategy based on their size combined with a specific inflammatory marker (ICAM-1) and to unravel their proteome signature and functional integrity at the onset of vascular inflammation. Here, we report that vascular endothelial cells upon inflammation release two heterogeneous size-based populations of EV (EV-10 K and EV-110 K) sharing a cocktail of inflammatory proteins, chemokines, and cytokines (chiefly: ICAM-1, CCL-2, CCL-4, CCL-5, IL-8 and CXCL-10). The co-enrichment of ICAM-1 and classical EV markers within these two size-based populations gave us a promising opportunity to further separate the inflammation-associated EV subpopulations, using an immuno-affinity methodology. Protein profiling of EV subpopulations highlighted that the phenotypic state of inflamed endothelial cells is preferentially mirrored in secreted medium- and large-sized ICAM-1 (+) EV. As functional players, the smaller-sized EV and especially their ICAM-1 (+) EV subpopulation promote the migration of THP-1 monocytes, whereas the large ICAM-1 (+) EV were more potent to induce ICAM-1 expression in recipient endothelial cells. This study provides new insights into the immunomodulatory content of inflammation-associated EV (sub)populations and their functional contributions to the initiation of vascular inflammation (ICAM-1 expression) and monocyte mobilization.
RESUMEN
The importance of vascular endothelial growth factor-A (VEGF) and subsequent Notch signaling in cardiac outflow tract development is generally recognized. Although genetic heterogeneity and mutations of these genes in both humans and mouse models relate to a high susceptibility to develop outflow tract malformations such as tetralogy of Fallot and peripheral pulmonary stenosis, no etiology has been proposed so far. Using immunohistochemistry, in situ hybridization, and quantitative RT-PCR on embryonic hearts, we have shown spatiotemporal increase and abnormal patterning of Vegf/VEGF/(phosphorylated) VEGFR-2, (cleaved) Notch1, and Jagged2 in the outflow tract of Vegf120/120 mouse embryos. This coincides with hyperplasia of specifically the outflow tract cushions and a high degree of subpulmonary myocardial apoptosis that, in later stages, manifest as pulmonary stenosis and ventricular septal defects. We postulate that increase of VEGF and Notch signaling during right ventricular outflow tract development can lead to abnormal development of both cushion and myocardial structures. Defective right ventricular outflow tract development as presented provides new insight in the etiology of tetralogy of Fallot.
Asunto(s)
Embrión de Mamíferos/anomalías , Miocardio/metabolismo , Receptor Notch1/genética , Transducción de Señal/genética , Tetralogía de Fallot/genética , Factor A de Crecimiento Endotelial Vascular/genética , Animales , Aorta Torácica/anomalías , Aorta Torácica/patología , Modelos Animales de Enfermedad , Embrión de Mamíferos/metabolismo , Embrión de Mamíferos/patología , Regulación del Desarrollo de la Expresión Génica , Ventrículos Cardíacos/anomalías , Ventrículos Cardíacos/patología , Inmunohistoquímica , Hibridación in Situ , Proteína Jagged-2 , Proteínas de la Membrana/metabolismo , Ratones , Ratones Mutantes , Miocardio/patología , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , ARN Mensajero/metabolismo , Receptor Notch1/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor A de Crecimiento Endotelial Vascular/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
AIMS: Currently, many potential cardiac revascularization therapies target the vascular endothelial growth factor (VEGF) pathway, with variable success. Knowledge regarding the role of the VEGF/Notch/ephrinB2 cascade in (ab)normal coronary development will provide information on the subtle balance of VEGF signalling in coronary maturation and might enhance our therapeutic possibilities. METHODS AND RESULTS: The effect of VEGF isoforms on coronary development was explored in vivo using immunohistochemistry and RT-qPCR on Vegf120/120 mouse embryos solely expressing VEGF120. In vitro, human arterial coronary endothelial cells were treated with VEGF121 or VEGF165 upon which RT-qPCR was performed. In vivo, mutant coronary arterial endothelium showed a decrease in protein expression of arterial markers such as cleaved Notch1, Delta-like4, and ephrinB2 concomitant with an increase of venous markers such as chicken ovalbumin upstream promoter transcription factor II. The venous endothelium showed the opposite effect, which was confirmed on the mRNA level. In vitro, mRNA expression of arterial markers highly depended on the VEGF isoform used, with VEGF165 having the strongest effect. Also, coronary arteriogenesis was anomalous in the mouse embryos with decreased arterial and increased venous medial development as shown by staining for smooth muscle alpha-actin, Delta-like1, and Notch3. CONCLUSION: We demonstrate that VEGF isoform-related spatiotemporal cardiac alterations in the VEGF/Notch/ephrinB2 cascade lead to disturbed coronary development. This knowledge can contribute to optimizing therapies targeting VEGF signalling by enabling balancing between angiogenesis and vascular maturation.
Asunto(s)
Vasos Coronarios/metabolismo , Corazón/embriología , Miocardio/metabolismo , Neovascularización Fisiológica , Receptores Notch/metabolismo , Transducción de Señal , Factores de Crecimiento Endotelial Vascular/metabolismo , Animales , Diferenciación Celular , Células Cultivadas , Vasos Coronarios/embriología , Células Endoteliales/metabolismo , Efrina-B2/metabolismo , Regulación del Desarrollo de la Expresión Génica , Humanos , Inmunohistoquímica , Ratones , Ratones Transgénicos , Miocardio/patología , Neovascularización Fisiológica/genética , Isoformas de Proteínas/metabolismo , ARN Mensajero/metabolismo , Receptores Notch/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/genética , Factores de Tiempo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Factores de Crecimiento Endotelial Vascular/genética , Venas/embriología , Venas/metabolismoRESUMEN
Given the major structural role phosphodiesters play in the organism it is surprising they have not been more widely adopted as a building block in sophisticated biomimetic hydrogels and other biomaterials. The potential benefits are substantial: phosphoester-based materials show excellent compatibility with blood, cells, and a remarkable resistance to protein adsorption that may trigger a foreign-body response. In this work, a novel class of phosphodiester-based ionic hydrogels is presented which are crosslinked via a phosphodiester moiety. The material shows good compatibility with blood, supports the growth and proliferation of tissue and presents opportunities for use as a drug release matrix as shown with fluorescent model compounds. The final gel is produced via base-induced elimination from a phosphotriester precursor, which is made by the free-radical polymerization of a phosphotriester crosslinker. This crosslinker is easily synthesized via multigram one-pot procedures out of common laboratory chemicals. Via the addition of various comonomers the properties of the final gel may be tuned leading to a wide range of novel applications for this exciting class of materials.
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Liberación de Fármacos , Ésteres/química , Hidrogeles/química , Andamios del Tejido/química , Animales , Dimetilsulfóxido/química , Colorantes Fluorescentes/química , Colorantes Fluorescentes/metabolismo , Liofilización , Espectroscopía de Resonancia Magnética , Ensayo de Materiales , Miocitos del Músculo Liso/citología , PorcinosRESUMEN
Autologous fat transfer (AFT) is limited by post-operative volume loss due to ischemia-induced cell death in the fat graft. Previous studies have demonstrated that electrical stimulation (ES) promotes angiogenesis in a variety of tissues and cell types. In this study we investigated the effects of ES on the angiogenic potential of adipose-derived stem cells (ASC), important progenitor cells in fat grafts with proven angiogenic potential. Cultured human ASC were electrically stimulated for 72 hours after which the medium of stimulated (ES) and non-stimulated (control) ASC was analysed for angiogenesis-related proteins by protein array and ELISA. The functional effect of ES on angiogenesis was then assessed in vitro and in vivo. Nine angiogenesis-related proteins were detected in the medium of electrically (non-)stimulated ASC and were quantified by ELISA. The pro-angiogenic proteins VEGF and MCP-1 were significantly increased following ES compared to controls, while the anti-angiogenic factor Serpin E1/PAI-1 was significantly decreased. Despite increased levels of anti-angiogenic TSP-1 and TIMP-1, medium of ES-treated ASC significantly increased vessel density, total vessel network length and branching points in chorio-allantoic membrane assays. In conclusion, our proof-of-concept study showed that ES increased the angiogenic potential of ASC both in vitro and in vivo.
Asunto(s)
Células Madre Mesenquimatosas/citología , Morfogénesis/efectos de la radiación , Neovascularización Fisiológica/efectos de la radiación , Trasplantes/crecimiento & desarrollo , Adipocitos/efectos de la radiación , Animales , Apoptosis/genética , Apoptosis/efectos de la radiación , Diferenciación Celular/efectos de la radiación , Células Cultivadas , Embrión de Pollo , Medios de Cultivo Condicionados/farmacología , Estimulación Eléctrica , Regulación del Desarrollo de la Expresión Génica/efectos de la radiación , Humanos , Células Madre Mesenquimatosas/efectos de la radiación , Morfogénesis/genética , Neovascularización Fisiológica/fisiología , Células Madre/efectos de la radiación , Trasplantes/efectos de la radiaciónRESUMEN
Extracellular vesicles (EV) mediated intercellular communication between monocytes and endothelial cells (EC) might play a major role in vascular inflammation and atherosclerotic plaque formation during cardiovascular diseases (CVD). While critical involvement of small (exosomes) and large EV (microvesicles) in CVD has recently been appreciated, the pro- and/or anti-inflammatory impact of a bulk EV (exosomes + microvesicles) on vascular cell function as well as their inflammatory capacity are poorly defined. This study aims to unravel the immunomodulatory content of EV bulk derived from control (uEV) and TNF-α induced inflamed endothelial cells (tEV) and to define their capacity to affect the inflammatory status of recipients monocytes (THP-1) and endothelial cells (HUVEC) in vitro. Here, we show that EV derived from inflamed vascular EC were readily taken up by THP-1 and HUVEC. Human inflammation antibody array together with ELISA revealed that tEV contain a pro-inflammatory profile with chemotactic mediators, including intercellular adhesion molecule (ICAM)-1, CCL-2, IL-6, IL-8, CXCL-10, CCL-5, and TNF-α as compared to uEV. In addition, EV may mediate a selective transfer of functional inflammatory mediators to their target cells and modulate them toward either pro-inflammatory (HUVEC) or anti/pro-inflammatory (THP-1) mode. Accordingly, the expression of pro-inflammatory markers (IL-6, IL-8, and ICAM-1) in tEV-treated HUVEC was increased. In the case of THP-1, EC-EV do induce a mixed of pro- and anti-inflammatory response as indicated by the elevated expression of ICAM-1, CCL-4, CCL-5, and CXCL-10 proteins. At the functional level, EC-EV mediated inflammation and promoted the adhesion and migration of THP-1. Taken together, our findings proved that the EV released from inflamed EC were enriched with a cocktail of inflammatory markers, chemokines, and cytokines which are able to establish a targeted cross-talk between EC and monocytes and reprogramming them toward a pro- or anti-inflammatory phenotypes.
Asunto(s)
Células Endoteliales/metabolismo , Vesículas Extracelulares/metabolismo , Mediadores de Inflamación/metabolismo , Monocitos/metabolismo , Quimiocinas/metabolismo , Citocinas/metabolismo , Células Endoteliales de la Vena Umbilical Humana , Humanos , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Isolating and maintaining the appropriate stem cell for large scale cell culture is essential in tissue engineering or food production. For bovine satellite cells an optimized isolation and purification protocol is lacking and there is also no detailed understanding on the factors that maintain stemness of these cells. Here, we set up a fluorescence-activated cell sorting strategy to enrich bovine satellite cells. We found that p38-MAPK signalling is activated and PAX7 expression is gradually lost during satellite cell proliferation. The p38 inhibitor (SB203580) treatment maintained PAX7 expression but inhibited the fusion of satellite cells in a concentration-dependent way in short-term incubation. The mechanism of p38 inhibition was confirmed by inhibiting canonical p38 signalling, i.e. HSP27. Long-term culture with an appropriate concentration of p38i enhanced the proliferation and PAX7 expression, while the differentiation capacity recovered and was enhanced compared to vehicle control. These studies indicate that bovine satellite cells maintenance depends on cell purity and p38 MAPK signalling. Inhibition of p38 MAPK signaling is a promising strategy to facilitate large scale cell expansion of primary cells for tissue engineering and cultured meat purposes.