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Šimák, Jan; Holada, Karel; Vostal, Jaroslav G.

doi:10.1186/1471-2121-3-11

Cited by 78 publications

(11 citation statements)

References 32 publications

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“…[15–17] Pretreatment of HPAEC with Rho kinase inhibitor Y-27632 or calpain inhibitor calpeptin did not alter CS-induced MP production, but this effect was suppressed by pan-caspase inhibitor Z-VAD-FMK (Fig. 2A).…”

Section: Resultsmentioning

confidence: 99%

Mechanical Forces Stimulate Endothelial Microparticle Generation via Caspase‐Dependent Apoptosis‐Independent Mechanism

et al. 2013

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Microparticle release by vascular endothelium has been implicated in various cardiovascular pathologies. Ventilator-induced lung injury (VILI) is a life-threatening complication of mechanical ventilation at high tidal volumes associated with excessive mechanical stretch of pulmonary vascular endothelial cells. However, a role of VILI-relevant levels of cyclic stretch in microparticle generation by vascular endothelium remains unknown. We report microparticle formation by human pulmonary endothelial cells exposed to pathologic, but not physiologic, levels of mechanical stress. Stretch-induced microparticle generation was not affected by cell co-treatment with inflammatory agents thrombin or bacterial wall lipopolysacharide. Neither the basal nor the pathologic cyclic stretch-induced microparticle production was affected by Rho kinase and calpain inhibitors, but were instead abolished by caspase inhibitor. In contrast to lipopolysacharide, pathologic mechanical strain did not significantly induce apoptosis in pulmonary endothelial cells. These results show for the first time that mechanical strain of pulmonary endothelial cells at levels relevant to high tidal volume mechanical ventilation is a potent activator of microparticle formation, which requires caspase activity; however, this mechanism is independent of apoptosis. These results suggest a novel mechanism that may contribute to VILI-associated vascular dysfunction.

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Section: Resultsmentioning

confidence: 99%

Mechanical Forces Stimulate Endothelial Microparticle Generation via Caspase‐Dependent Apoptosis‐Independent Mechanism

et al. 2013

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“…macrophage migration inhibitory factor), to name just a few. Short-term PMA treatment does not release microparticles (30), as shedding events make it difficult to discern intracellular from secreted/membrane proteins. In a direct comparison of the cellular proteome and the secretome utilizing difference gel electrophoresis, 70 out of 96 proteins analyzed were present in both samples, representing <10% of the visible protein spots in the secretome.…”

Section: Discussionmentioning

confidence: 99%

Glycoproteomic Analysis of the Secretome of Human Endothelial Cells

Yin

Bern

Xing

et al. 2013

Molecular & Cellular Proteomics

108

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Previous proteomics studies have partially unraveled the complexity of endothelial protein secretion but have not investigated glycosylation, a key modification of secreted and membrane proteins for cell communication. In this study, human umbilical vein endothelial cells were kept in serum-free medium before activation by phorbol-12-myristate-13 acetate, a commonly used secretagogue that induces exocytosis of endothelial vesicles. In addition to 123 secreted proteins, the secretome was particularly rich in membrane proteins. Glycopeptides were enriched by zwitterionic hydrophilic interaction liquid chromatography resins and were either treated with PNGase F and H218O or directly analyzed using a recently developed workflow combining higher-energy C-trap dissociation (HCD) with electron-transfer dissociation (ETD) for a hybrid linear ion trap–orbitrap mass spectrometer. After deglycosylation with PNGase F in the presence of H218O, 123 unique peptides displayed 18O-deamidation of asparagine, corresponding to 86 proteins with a total of 121 glycosylation sites. Direct glycopeptide analysis via HCD-ETD identified 131 glycopeptides from 59 proteins and 118 glycosylation sites, of which 41 were known, 51 were predicted, and 26 were novel. Two methods were compared: alternating HCD-ETD and HCD-product-dependent ETD. The former detected predominantly high-intensity, multiply charged glycopeptides, whereas the latter preferentially selected precursors with complex/hybrid glycans for fragmentation. Validation was performed by means of glycoprotein enrichment and analysis of the input, the flow-through, and the bound fraction. This study represents the most comprehensive characterization of endothelial protein secretion to date and demonstrates the potential of new HCD-ETD workflows for determining the glycosylation status of complex biological samples.

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“…Subsequently, EGM-2 growth media was supplemented with NDs suspension (25 μg/mL) in PBS previously sonicated for 20 min, to study NDs biocompatibility by evaluating the expression of apoptotic genes. As a positive control, camptothecin solution (5 μM) in DMSO/PBS was included to the EGM-2 growth media to induce cell apoptosis [24]. After incubation for 24 h, cells were lysed and apoptotic genes expression was analyzed using the RT-qPCR procedure.…”

Section: Methodsmentioning

confidence: 99%

Nanodiamond-based injectable hydrogel for sustained growth factor release: Preparation, characterization and in vitro analysis

et al. 2017

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Nanodiamonds (NDs) represent an emerging class of carbon nanomaterials that possess favorable physical and chemical properties to be used as multifunctional carriers for a variety of bioactive molecules. Here we report the synthesis and characterization of a new injectable ND-based nanocomposite hydrogel which facilitates a controlled release of therapeutic molecules for regenerative applications. In particular, we have formulated a thermosensitive hydrogel using gelatin, chitosan and NDs that provides a sustained release of exogenous human vascular endothelial growth factor (VEGF) for wound healing applications. Addition of NDs improved the mechanical properties of the injectable hydrogels without affecting its thermosensitive gelation properties. Biocompatibility of the generated hydrogel was verified by in vitro assessment of apoptotic gene expressions and anti-inflammatory interleukin productions. NDs were complexed with VEGF and the inclusion of this complex in the hydrogel network enabled the sustained release of the angiogenic growth factor. These results suggest for the first time that NDs can be used to formulate a biocompatible, thermosensitive and multifunctional hydrogel platform that can function both as a filling agent to modulate hydrogel properties, as well as a delivery platform for the controlled release of bioactive molecules and growth factors. Statement of Significance One of the major drawbacks associated with the use of conventional hydrogels as carriers of growth factors is their inability to control the release kinetics of the loaded molecules. In fact, in most cases, a burst release is inevitable leading to diminished therapeutic effects and unsuccessful therapies. As a potential solution to this issue, we hereby propose a strategy of incorporating ND complexes within an injectable hydrogel matrix. The functional groups on the surface of the NDs can establish interactions with the model growth factor VEGF and promote a prolonged release from the polymer network, therefore, providing a longer therapeutic effect. Our strategy demonstrates the efficacy of using NDs as an essential component for the design of a novel injectable nanocomposite system with improved release capabilities.

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Untitled

Cited by 78 publications

References 32 publications

Mechanical Forces Stimulate Endothelial Microparticle Generation via Caspase‐Dependent Apoptosis‐Independent Mechanism

Mechanical Forces Stimulate Endothelial Microparticle Generation via Caspase‐Dependent Apoptosis‐Independent Mechanism

Glycoproteomic Analysis of the Secretome of Human Endothelial Cells

Nanodiamond-based injectable hydrogel for sustained growth factor release: Preparation, characterization and in vitro analysis

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