In intravenous immunoglobulins (IVIG), and some other immunoglobulin products, protein particles have been implicated in adverse events. Role and mechanisms of immunoglobulin particles in vascular adverse effects of blood components and manufactured biologics have not been elucidated. We have developed a model of spherical silica microparticles (SiMPs) of distinct sizes 200-2000 nm coated with different IVIG- or albumin (HSA)-coronas and investigated their effects on cultured human umbilical vein endothelial cells (HUVEC). IVIG products (1-20 mg/mL), bare SiMPs or SiMPs with IVIG-corona, did not display significant toxicity to unstimulated HUVEC. In contrast, in TNFα-stimulated HUVEC, IVIG-SiMPs induced decrease of HUVEC viability compared to HSA-SiMPs, while no toxicity of soluble IVIG was observed. 200 nm IVIG-SiMPs after 24 h treatment further increased ICAM1 (intercellular adhesion molecule 1) and tissue factor surface expression, apoptosis, mammalian target of rapamacin (mTOR)-dependent activation of autophagy, and release of extracellular vesicles, positive for mitophagy markers. Toxic effects of IVIG-SiMPs were most prominent for 200 nm SiMPs and decreased with larger SiMP size. Using blocking antibodies, toxicity of IVIG-SiMPs was found dependent on FcγRII receptor expression on HUVEC, which increased after TNFα-stimulation. Similar results were observed with different IVIG products and research grade IgG preparations. In conclusion, submicron particles with immunoglobulin corona induced size-dependent toxicity in TNFα-stimulated HUVEC via FcγRII receptors, associated with apoptosis and mTOR-dependent activation of autophagy. Testing of IVIG toxicity in endothelial cells prestimulated with proinflammatory cytokines is relevant to clinical conditions. Our results warrant further studies on endothelial toxicity of sub-visible immunoglobulin particles.

• MeSH
• apoptóza účinky léků   MeSH
• autofagie * účinky léků   MeSH
• endoteliální buňky pupečníkové žíly (lidské) * účinky léků   metabolismus   MeSH
• intravenózní imunoglobuliny * MeSH
• lidé MeSH
• mezibuněčná adhezivní molekula-1 metabolismus   MeSH
• oxid křemičitý chemie   toxicita   MeSH
• proteinová korona metabolismus   MeSH
• receptory IgG * metabolismus   MeSH
• TNF-alfa * metabolismus   MeSH
• TOR serin-threoninkinasy metabolismus   MeSH
• velikost částic MeSH
• viabilita buněk účinky léků   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Metals are widely utilized as implant materials for bone fixtures as well as stents. Biodegradable versions of these implants are highly desirable since patients do not have to undergo a second surgery for the materials to be removed. Attractive options for such materials are zinc silver alloys since they also offer the benefit of being antibacterial. However, it is important to investigate the effect of the degradation products of such alloys on the surrounding cells, taking into account silver cytotoxicity. Here we investigated zinc alloyed with 1 % of silver (Zn1Ag) and how differently concentrated extracts (1 %-100 %) of this material impact human umbilical vein endothelial cells (HUVECs). More specifically, we focused on free radical generation and oxidative stress as well as the impact on cell viability. To determine free radical production we used diamond-based quantum sensing as well as conventional fluorescent assays. The viability was assessed by observing cell morphology and the metabolic activity via the MTT assay. We found that 1 % and 10 % extracts are well tolerated by the cells. However, at higher extract concentrations we observed severe impact on cell viability and oxidative stress. We were also able to show that quantum sensing was able to detect significant free radical generation even at the lowest tested concentrations.

• MeSH
• biokompatibilní materiály chemie   farmakologie   MeSH
• endoteliální buňky pupečníkové žíly (lidské) * účinky léků   MeSH
• lidé MeSH
• nanodiamanty * chemie   MeSH
• oxidační stres * účinky léků   MeSH
• slitiny * chemie   MeSH
• stříbro toxicita   chemie   MeSH
• testování materiálů metody   MeSH
• viabilita buněk * účinky léků   MeSH
• volné radikály metabolismus   MeSH
• vstřebatelné implantáty škodlivé účinky   MeSH
• zinek * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

The placenta plays a critical role in maternal-fetal nutrient transport and fetal protection against drugs. Creating physiological in vitro models to study these processes is crucial, but technically challenging. This study introduces an efficient cell model that mimics the human placental barrier using co-cultures of primary trophoblasts and primary human umbilical vein endothelial cells (HUVEC) on a Transwell®-based system. Monolayer formation was examined over 7 days by determining transepithelial electrical resistance (TEER), permeability of Lucifer yellow (LY) and inulin, localization of transport proteins at the trophoblast membrane (immunofluorescence), and syncytialization markers (RT-qPCR/ELISA). We analysed diffusion-based (caffeine/antipyrine) and transport-based (leucine/Rhodamine-123) processes to study the transfer of physiologically relevant compounds. The latter relies on the adequate localization and function of the amino-acid transporter LAT1 and the drug transporter P-glycoprotein (P-gp) which were studied by immunofluorescence microscopy and application of respective inhibitors (2-Amino-2-norbornanecarboxylic acid (BCH) for LAT1; cyclosporine-A for P-gp). The formation of functional monolayer(s) was confirmed by increasing TEER values, low LY transfer rates, minimal inulin leakage, and appropriate expression/release of syncytialization markers. These results were supported by microscopic monitoring of monolayer formation. LAT1 was identified on the apical and basal sides of the trophoblast monolayer, while P-gp was apically localized. Transport assays confirmed the inhibition of LAT1 by BCH, reducing both intracellular leucine levels and leucine transport to the basal compartment. Inhibiting P-gp with cyclosporine-A increased intracellular Rhodamine-123 concentrations. Our in vitro model mimics key aspects of the human placental barrier. It represents a powerful tool to study nutrient and drug transport mechanisms across the placenta, assisting in evaluating safer pregnancy therapies.

• MeSH
• biologické modely MeSH
• biologický transport MeSH
• endoteliální buňky pupečníkové žíly (lidské) * metabolismus   MeSH
• inulin metabolismus   MeSH
• isochinoliny MeSH
• kokultivační techniky MeSH
• leucin metabolismus   MeSH
• lidé MeSH
• maternofetální výměna látek * MeSH
• P-glykoprotein metabolismus   MeSH
• placenta * metabolismus   MeSH
• rhodamin 123 metabolismus   MeSH
• těhotenství MeSH
• trofoblasty * metabolismus   MeSH
• Check Tag
• lidé MeSH
• těhotenství MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH

Long-term delivery of growth factors and immunomodulatory agents is highly required to support the integrity of tissue in engineering constructs, e.g., formation of vasculature, and to minimize immune response in a recipient. However, for proteins with a net positive charge at the physiological pH, controlled delivery from negatively charged alginate (Alg) platforms is challenging due to electrostatic interactions that can hamper the protein release. In order to regulate such interactions between proteins and the Alg matrix, we propose to complex proteins of interest in this study - CXCL12, FGF-2, VEGF - with polyanionic heparin prior to their encapsulation into Alg microbeads of high content of α-L-guluronic acid units (high-G). This strategy effectively reduced protein interactions with Alg (as shown by model ITC and SPR experiments) and, depending on the protein type, afforded control over the protein release for at least one month. The released proteins retained their in vitro bioactivity: CXCL12 stimulated the migration of Jurkat cells, and FGF-2 and VEGF induced proliferation and maturation of HUVECs. The presence of heparin also intensified protein biological efficiency. The proposed approach for encapsulation of proteins with a positive net charge into high-G Alg hydrogels is promising for controlled long-term protein delivery under in vivo conditions.

• MeSH
• algináty chemie   MeSH
• chemokin CXCL12 chemie   MeSH
• endoteliální buňky pupečníkové žíly (lidské) MeSH
• fibroblastový růstový faktor 2 chemie   MeSH
• heparin chemie   MeSH
• lidé MeSH
• mikrosféry MeSH
• nádorové buněčné linie MeSH
• tkáňové inženýrství MeSH
• vaskulární endoteliální růstový faktor A chemie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH

Efflux transporters, namely ATP-binding cassette (ABC), are one of the primary reasons for cancer chemoresistance and the clinical failure of chemotherapy. Ganciclovir (GCV) is an antiviral agent used in herpes simplex virus thymidine kinase (HSV-TK) gene therapy. In this therapy, HSV-TK gene is delivered together with GCV into cancer cells to activate the phosphorylation process of GCV to active GCV-triphosphate, a DNA polymerase inhibitor. However, GCV interacts with efflux transporters that are responsible for the resistance of HSV-TK/GCV therapy. In the present study, it was explored whether GCV and its more lipophilic derivative (1) could inhibit effluxing of another chemotherapeutic, methotrexate (MTX), out of the human breast cancer cells. Firstly, it was found that the combination of GCV and MTX was more hemocompatible than the corresponding combination with compound 1. Secondly, both GCV and compound 1 enhanced the cellular accumulation of MTX in MCF-7 cells, the MTX exposure being 13-21 times greater compared to the MTX uptake alone. Subsequently, this also reduced the number of viable cells (41-56%) and increased the number of late apoptotic cells (46-55%). Moreover, both GCV and compound 1 were found to interact with breast cancer resistant protein (BCRP) more effectively than multidrug-resistant proteins (MRPs) in these cells. Since the expression of BCRP was higher in MCF-7 cells than in MDA-MB-231 cells, and the cellular uptake of GCV and compound 1 was smaller but increased in the presence of BCRP-selective inhibitor (Fumitremorgin C) in MCF-7 cells, we concluded that the improved apoptotic effects of higher MTX exposure were raised mainly from the inhibition of BCRP-mediated efflux of MTX. However, the effects of GCV and its derivatives on MTX metabolism and the quantitative expression of MTX metabolizing enzymes in various cancer cells need to be studied more thoroughly in the future.

• MeSH
• ABC transportér z rodiny G, člen 2 metabolismus   MeSH
• antivirové látky farmakologie   MeSH
• apoptóza účinky léků   MeSH
• endoteliální buňky pupečníkové žíly (lidské) MeSH
• ganciklovir farmakologie   MeSH
• lidé MeSH
• methotrexát farmakologie   MeSH
• MFC-7 buňky MeSH
• nádorové buněčné linie MeSH
• nádorové proteiny metabolismus   MeSH
• nádory prsu farmakoterapie   metabolismus   MeSH
• proteiny spojené s mnohočetnou rezistencí k lékům metabolismus   MeSH
• Check Tag
• lidé MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH

Iron is essential for a healthy pregnancy, and iron supplementation is nearly universally recommended, regardless of maternal iron status. A signal of potential harm is the U-shaped association between maternal ferritin, a marker of iron stores, and risk of adverse pregnancy outcomes. However, ferritin is also induced by inflammation and may overestimate iron stores during inflammation or infection. In this study, we use mouse models to determine whether maternal iron loading, inflammation, or their interaction cause poor pregnancy outcomes. Only maternal exposure to both iron excess and inflammation, but not either condition alone, causes embryo malformations and demise. Maternal iron excess potentiates embryo injury during both LPS-induced acute inflammation and obesity-induced chronic mild inflammation. The adverse interaction depends on TNFα signaling, causes apoptosis of placental and embryo endothelium, and is prevented by anti-TNFα or antioxidant treatment. Our findings raise important questions about the safety of indiscriminate iron supplementation during pregnancy.

• MeSH
• apoptóza fyziologie   MeSH
• embryo savčí patologie   MeSH
• endoteliální buňky pupečníkové žíly (lidské) MeSH
• ferritin analýza   MeSH
• hepcidiny genetika   MeSH
• komplikace těhotenství MeSH
• kultivované buňky MeSH
• lidé MeSH
• myši inbrední C57BL MeSH
• myši knockoutované MeSH
• myši MeSH
• obezita patologie   MeSH
• placenta patologie   MeSH
• těhotenství MeSH
• TNF-alfa metabolismus   MeSH
• železo metabolismus   toxicita   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• těhotenství MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH

Galectin-3 (Gal-3) is a β-galactoside-binding protein that influences various cell functions, including cell adhesion. We focused on the role of Gal-3 as an extracellular ligand mediating cell-matrix adhesion. We used human adipose tissue-derived stem cells and human umbilical vein endothelial cells that are promising for vascular tissue engineering. We found that these cells naturally contained Gal-3 on their surface and inside the cells. Moreover, they were able to associate with exogenous Gal-3 added to the culture medium. This association was reduced with a β-galactoside LacdiNAc (GalNAcβ1,4GlcNAc), a selective ligand of Gal-3, which binds to the carbohydrate recognition domain (CRD) in the Gal-3 molecule. This ligand was also able to detach Gal-3 newly associated with cells but not Gal-3 naturally present on cells. In addition, Gal-3 preadsorbed on plastic surfaces acted as an adhesion ligand for both cell types, and the cell adhesion was resistant to blocking with LacdiNAc. This result suggests that the adhesion was mediated by a binding site different from the CRD. The blocking of integrin adhesion receptors on cells with specific antibodies revealed that the cell adhesion to the preadsorbed Gal-3 was mediated, at least partially, by β1 and αV integrins-namely α5β1, αVβ3, and αVβ1 integrins.

• MeSH
• buněčná adheze * MeSH
• endoteliální buňky pupečníkové žíly (lidské) cytologie   fyziologie   MeSH
• galektiny metabolismus   MeSH
• integriny metabolismus   MeSH
• krevní proteiny metabolismus   MeSH
• kultivované buňky MeSH
• lidé MeSH
• mezenchymální kmenové buňky cytologie   fyziologie   MeSH
• spoje buňka-matrix metabolismus   MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Most therapeutic agents used for treating brain malignancies face hindered transport through the blood-brain barrier (BBB) and poor tissue penetration. To overcome these problems, we developed peptide conjugates of conventional and experimental anticancer agents. SynB3 cell-penetrating peptide derivatives were applied that can cross the BBB. Tuftsin derivatives were used to target the neuropilin-1 transport system for selectivity and better tumor penetration. Moreover, SynB3-tuftsin tandem compounds were synthesized to combine the beneficial properties of these peptides. Most of the conjugates showed high and selective efficacy against glioblastoma cells. SynB3 and tandem derivatives demonstrated superior cellular internalization. The penetration profile of the conjugates was determined on a lipid monolayer and Transwell co-culture system with noncontact HUVEC-U87 monolayers as simple ex vivo and in vitro BBB models. Importantly, in 3D spheroids, daunomycin-peptide conjugates possessed a better tumor penetration ability than daunomycin. These conjugates are promising tools for the delivery systems with tunable features.

• MeSH
• antitumorózní látky chemie   farmakokinetika   farmakologie   MeSH
• buněčné sféroidy účinky léků   metabolismus   MeSH
• endoteliální buňky pupečníkové žíly (lidské) MeSH
• glioblastom farmakoterapie   metabolismus   MeSH
• hematoencefalická bariéra metabolismus   MeSH
• krysa rodu rattus MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• nádorové buňky kultivované MeSH
• nádory mozku farmakoterapie   metabolismus   MeSH
• neuropilin-1 metabolismus   MeSH
• nosiče léků chemie   farmakokinetika   farmakologie   MeSH
• oligopeptidy chemie   farmakokinetika   farmakologie   MeSH
• penetrační peptidy chemie   farmakokinetika   farmakologie   MeSH
• systémy cílené aplikace léků MeSH
• tuftsin analogy a deriváty   farmakokinetika   farmakologie   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Pulmonary arterial hypertension (PAH) isa fatal disease characterized by vascular remodeling and chronic inflammation. Macrophages are the key orchestrators of inflammatory and repair responses, and have been demonstrated to be vital in the pathogenesis of PAH. However, specific phenotype of macrophage polarization (M1 & M2 macrophage) in the development of PAH and the underlying mechanisms how they work are still largely unclear. A rat model of monocrotaline (MCT) induced PAH was used. Hemodynamic analysis and histopathological experiments were conducted at day 3, 7, 14, 21 and 28, respectively. In PAH rat lung tissue, confocal microscopic images showed that CD68+NOS2+ M1-like macrophages were remarkably infiltrated on early stage, but dramatically decreased in mid-late stage. Meanwhile, CD68+CD206+ M2-like macrophages in lung tissue accumulated gradually since day 7 to day 28, and the relative ratio of M2/M1 macrophage increased over time. Results detected by western blot and immunohistochemistry were consistent. Further vitro functional studies revealed the possible mechanism involved in this pathophysiological process. By using Transwell co-culture system, it was found that M1 macrophages inducedendothelial cellapoptosis, while M2 macrophages significantly promoted proliferation of both endothelial cell and smooth muscle cell.These data preliminarily demonstrated a temporal dynamic change of macrophage M1/M2 polarization status in the development of experimental PAH. M1 macrophages participated in the initial stage of inflammation by accelerating apoptosis of endothelial cell, while M2 macrophages predominated in the reparative stage of inflammation and the followed stage of aberrant tissue remodeling.

• MeSH
• antigeny diferenciační myelomonocytární metabolismus   MeSH
• apoptóza MeSH
• arteria pulmonalis metabolismus   patologie   MeSH
• časové faktory MeSH
• CD antigeny metabolismus   MeSH
• cytokiny metabolismus   MeSH
• endoteliální buňky pupečníkové žíly (lidské) metabolismus   patologie   MeSH
• fenotyp MeSH
• kokultivační techniky MeSH
• kultivované buňky MeSH
• lidé MeSH
• makrofágy metabolismus   patologie   MeSH
• mediátory zánětu metabolismus   MeSH
• modely nemocí na zvířatech MeSH
• monokrotalin MeSH
• myocyty hladké svaloviny metabolismus   patologie   MeSH
• plicní arteriální hypertenze chemicky indukované   metabolismus   patologie   MeSH
• potkani Sprague-Dawley MeSH
• proliferace buněk MeSH
• receptor mannózy metabolismus   MeSH
• remodelace cév * MeSH
• synthasa oxidu dusnatého, typ II metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

An ideal decellularized allogenic or xenogeneic cardiovascular graft should be capable of preventing thrombus formation after implantation. The antithrombogenicity of the graft is ensured by a confluent endothelial cell layer formed on its surface. Later repopulation and remodeling of the scaffold by the patient's cells should result in the formation of living autologous tissue. In the work presented here, decellularized porcine pericardium scaffolds were modified by growing a fibrin mesh on the surface and inside the scaffolds, and by attaching heparin and human vascular endothelial growth factor (VEGF) to this mesh. Then the scaffolds were seeded with human adipose tissue-derived stem cells (ASCs). While the ASCs grew only on the surface of the decellularized pericardium, the fibrin-modified scaffolds were entirely repopulated in 28 d, and the scaffolds modified with fibrin, heparin and VEGF were already repopulated within 6 d. Label free mass spectrometry revealed fibronectin, collagens, and other extracellular matrix proteins produced by ASCs during recellularization. Thin layers of human umbilical endothelial cells were formed within 4 d after the cells were seeded on the surfaces of the scaffold, which had previously been seeded with ASCs. The results indicate that an artificial tissue prepared by in vitro recellularization and remodeling of decellularized non-autologous pericardium with autologous ASCs seems to be a promising candidate for cardiovascular grafts capable of accelerating in situ endothelialization. ASCs resemble the valve interstitial cells present in heart valves. An advantage of this approach is that ASCs can easily be collected from the patient by liposuction.

• MeSH
• bioprotézy MeSH
• decelularizovaná extracelulární matrix chemie   MeSH
• endoteliální buňky pupečníkové žíly (lidské) MeSH
• endoteliální buňky cytologie   MeSH
• extracelulární matrix metabolismus   MeSH
• fibrinogen chemie   MeSH
• fibronektiny chemie   MeSH
• fluorescenční mikroskopie MeSH
• kmenové buňky MeSH
• kolagen chemie   MeSH
• lidé MeSH
• lipektomie MeSH
• perikard metabolismus   patologie   MeSH
• prasata MeSH
• proliferace buněk MeSH
• srdeční chlopně * MeSH
• techniky in vitro MeSH
• thrombin chemie   MeSH
• tkáňové inženýrství metody   MeSH
• tkáňové podpůrné struktury * chemie   MeSH
• tuková tkáň cytologie   MeSH
• vaskulární endoteliální růstový faktor A metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH