The global shortage of corneal endothelial graft tissue necessitates the exploration of alternative therapeutic strategies. Rho-associated protein kinase inhibitors (ROCKi), recognized for their regenerative potential in cardiology, oncology, and neurology, have shown promise in corneal endothelial regeneration. This study investigates the repurposing potential of additional ROCKi compounds. Through screening a self-assembled library of ROCKi on B4G12 corneal endothelial cells, we evaluated their dose-dependent effects on proliferation, migration, and toxicity using live-cell imaging. Nine ROCKi candidates significantly enhanced B4G12 proliferation compared to the basal growth rate. These candidates were further assessed for their potential to accelerate wound closure as another indicator for tissue regeneration capacity, with most demonstrating notable efficacy. To assess the potential impact of candidate ROCKi on key corneal endothelial cell markers related to cell proliferation, leaky tight junctions and ion efflux capacity, we analyzed the protein expression of cyclin E1, CDK2, p16, ZO-1 and Na+/K+-ATPase, respectively. Immunocytochemistry and western blot analysis confirmed the preservation of corneal endothelial markers post-treatment with ROCKi hits. However, notable cytoplasm enlargement and nuclear fragmentation were detected after the treatment with SR-3677 and Thiazovivin, indicating possible cellular stress. In compared parameters, Chroman-1 at a concentration of 10 nM outperformed other ROCKi, requiring significantly 1000-fold lower effective concentration than established ROCKi Y-27632 and Fasudil. Altogether, this study underscores the potential of repurposing ROCKi for treating corneal endothelial dysfunctions, offering a viable alternative to conventional grafting methods, and highlights Chroman-1 as a promising candidate structure for hit-to-lead development.
- Klíčová slova
- Chroman-1, Corneal endothelial regeneration, Drug repurposing, ROCK inhibitors, Small molecule screening,
- MeSH
- buněčné linie MeSH
- endoteliální buňky účinky léků MeSH
- inhibitory proteinkinas * farmakologie MeSH
- kinázy asociované s rho * antagonisté a inhibitory metabolismus MeSH
- lidé MeSH
- pohyb buněk účinky léků MeSH
- přehodnocení terapeutických indikací léčivého přípravku MeSH
- proliferace buněk * účinky léků MeSH
- regenerace * účinky léků MeSH
- rohovkový endotel * účinky léků MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- inhibitory proteinkinas * MeSH
- kinázy asociované s rho * MeSH
Monocyte homing to the liver and adhesion to the liver sinusoidal endothelial cells (LSECs) are key elements in nonalcoholic steatohepatitis (NASH) pathogenesis. We reported previously that VCAM-1 mediates monocyte adhesion to LSECs. However, the pathogenic role of VCAM-1 in NASH is unclear. Herein, we report that VCAM-1 was a top upregulated adhesion molecule in the NASH mouse liver transcriptome. Open chromatin landscape profiling combined with genome-wide transcriptome analysis showed robust transcriptional upregulation of LSEC VCAM-1 in murine NASH. Moreover, LSEC VCAM-1 expression was significantly increased in human NASH. LSEC VCAM-1 expression was upregulated by palmitate treatment in vitro and reduced with inhibition of the mitogen-activated protein 3 kinase (MAP3K) mixed lineage kinase 3 (MLK3). Likewise, LSEC VCAM-1 expression was reduced in the Mlk3-/- mice with diet-induced NASH. Furthermore, VCAM-1 neutralizing Ab or pharmacological inhibition attenuated diet-induced NASH in mice, mainly via reducing the proinflammatory monocyte hepatic population as examined by mass cytometry by time of flight (CyTOF). Moreover, endothelium-specific Vcam1 knockout mice were also protected against NASH. In summary, lipotoxic stress enhances the expression of LSEC VCAM-1, in part, through MLK3 signaling. Inhibition of VCAM-1 was salutary in murine NASH and might serve as a potential therapeutic strategy for human NASH.
- Klíčová slova
- Cell migration/adhesion, Endothelial cells, Hepatology,
- MeSH
- cévní buněčněadhezivní molekula-1 antagonisté a inhibitory genetika metabolismus MeSH
- endoteliální buňky účinky léků metabolismus MeSH
- játra účinky léků metabolismus patologie MeSH
- lidé MeSH
- MAP kinasový signální systém účinky léků MeSH
- messenger RNA genetika MeSH
- modely nemocí na zvířatech MeSH
- myši inbrední C57BL MeSH
- myši knockoutované MeSH
- myši MeSH
- nealkoholová steatóza jater etiologie genetika metabolismus MeSH
- neutralizující protilátky aplikace a dávkování MeSH
- palmitany toxicita MeSH
- stanovení celkové genové exprese MeSH
- upregulace účinky léků MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
- Názvy látek
- cévní buněčněadhezivní molekula-1 MeSH
- messenger RNA MeSH
- neutralizující protilátky MeSH
- palmitany MeSH
Silicon dioxide, in the form of nanoparticles, possesses unique physicochemical properties (size, shape, and a large surface to volume ratio). Therefore, it is one of the most promising materials used in biomedicine. In this paper, we compare the biological effects of both mesoporous silica nanoparticles extracted from Urtica dioica L. and pyrogenic material. Both SEM and TEM investigations confirmed the size range of tested nanoparticles was between 6 and 20 nanometers and their amorphous structure. The cytotoxic activity of the compounds and intracellular ROS were determined in relation to cells HMEC-1 and erythrocytes. The cytotoxic effects of SiO2 NPs were determined after exposure to different concentrations and three periods of incubation. The same effects for endothelial cells were tested under the same range of concentrations but after 2 and 24 h of exposure to erythrocytes. The cell viability was measured using spectrophotometric and fluorimetric assays, and the impact of the nanoparticles on the level of intracellular ROS. The obtained results indicated that bioSiO2 NPs, present higher toxicity than pyrogenic NPs and have a higher influence on ROS production. Mesoporous silica nanoparticles show good hemocompatibility but after a 24 h incubation of erythrocytes with silica, the increase in hemolysis process, the decrease in osmotic resistance of red blood cells, and shape of erythrocytes changed were observed.
- Klíčová slova
- biogenic and pyrogenic silicon dioxide, cytotoxicity, mesoporous silica, nanoparticles,
- MeSH
- endoteliální buňky účinky léků MeSH
- erytrocyty účinky léků MeSH
- hemolýza účinky léků MeSH
- lidé MeSH
- nanočástice aplikace a dávkování chemie MeSH
- oxid křemičitý aplikace a dávkování chemie MeSH
- oxidační stres účinky léků MeSH
- poréznost MeSH
- povrchové vlastnosti MeSH
- reaktivní formy kyslíku metabolismus MeSH
- viabilita buněk MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- oxid křemičitý MeSH
- reaktivní formy kyslíku MeSH
BACKGROUND: The aim of our study was to assess the impact of different thawing protocols on morphological changes arising in cryopreserved human saphenous vein grafts. METHODS: The study was performed in 12 saphenous vein grafts harvested in brain death donors. Storage in the vapor phase of liquid nitrogen for 3 or 5 years followed. Two thawing protocols were tested: slow thawing in a refrigerator at temperature +4°C for 2 hr and rapid thawing-in a water bath at +37°C. Grafts were processed for scanning electron microscopy. Comparisons of continuous parameters under study between experimental groups were performed using the t-test (age, cold ischemia time, exposure to cryoprotectant, time of storage, total thawing time, mean thawing rate, morphology scoring of thawed HSVG) and the median test (HSVG length). Categorical parameters (sex and blood group) were formally tested using the chi-square test. RESULTS: All samples were evaluated according to morphological changes and scored in terms of morphologically intact endothelium, confluent endothelium with structural inhomogeneity, disruption of the intercellular contacts, separation of the endothelial cells, complete loss of the endothelium, and damage of the subendothelial layers. There is no statistically significant difference between the sample sets at the significance level of 0.05. There was no association with donors' age, sex, and time of storage. CONCLUSIONS: Human cryopreserved saphenous vein grafts in our experimental work showed no difference in terms of structural deterioration of the endothelial surface and basal membrane depending on different thawing protocols used.
- MeSH
- časové faktory MeSH
- dospělí MeSH
- endoteliální buňky účinky léků transplantace ultrastruktura MeSH
- kryoprezervace * MeSH
- kryoprotektivní látky farmakologie MeSH
- lidé středního věku MeSH
- lidé MeSH
- mladiství MeSH
- mladý dospělý MeSH
- odběr tkání a orgánů MeSH
- přežití tkáně MeSH
- vena saphena účinky léků transplantace ultrastruktura MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mladiství MeSH
- mladý dospělý MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- srovnávací studie MeSH
- Názvy látek
- kryoprotektivní látky MeSH
Arthrospira platensis, a blue-green alga, is a popular nutraceutical substance having potent antioxidant properties with potential anti-carcinogenic activities. The aim of our study was to assess the possible anti-angiogenic effects of A platensis in an experimental model of pancreatic cancer. The effects of an A platensis extract were investigated on human pancreatic cancer cells (PA-TU-8902) and immortalized endothelial-like cells (Ea.hy926). PA-TU-8902 pancreatic tumours xenografted to athymic mice were also examined. In vitro migration and invasiveness assays were performed on the tested cells. Multiple angiogenic factors and signalling pathways were analysed in the epithelial, endothelial and cancer cells, and tumour tissue. The A platensis extract exerted inhibitory effects on both migration and invasion of pancreatic cancer as well as endothelial-like cells. Tumours of mice treated with A platensis exhibited much lesser degrees of vascularization as measured by CD31 immunostaining (P = .004). Surprisingly, the VEGF-A mRNA and protein expressions were up-regulated in pancreatic cancer cells. A platensis inhibited ERK activation upstream of Raf and suppressed the expression of ERK-regulated proteins. Treatment of pancreatic cancer with A platensis was associated with suppressive effects on migration and invasiveness with various anti-angiogenic features, which might account for the anticancer effects of this blue-green alga.
- Klíčová slova
- Arthrospira platensis, angiogenesis, anticancer effects, carcinogenesis, pancreatic cancer,
- MeSH
- antioxidancia farmakologie MeSH
- antitumorózní látky farmakologie MeSH
- endoteliální buňky účinky léků MeSH
- inhibitory angiogeneze farmakologie MeSH
- lidé MeSH
- myši nahé MeSH
- myši MeSH
- nádorové buněčné linie MeSH
- nádory slinivky břišní farmakoterapie MeSH
- patologická angiogeneze farmakoterapie MeSH
- pohyb buněk účinky léků MeSH
- signální transdukce účinky léků MeSH
- Spirulina chemie MeSH
- upregulace účinky léků MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- antioxidancia MeSH
- antitumorózní látky MeSH
- inhibitory angiogeneze MeSH
INTRODUCTION: Shiga toxin 2a (Stx2a) induces hemolytic uremic syndrome (STEC HUS) by targeting glomerular endothelial cells (GEC). OBJECTIVES: We investigated in a metabolomic analysis the response of a conditionally immortalized, stable glomerular endothelial cell line (ciGEnC) to Stx2a stimulation as a cell culture model for STEC HUS. METHODS: CiGEnC were treated with tumor necrosis factor-(TNF)α, Stx2a or sequentially with TNFα and Stx2a. We performed a metabolomic high-throughput screening by lipid- or gas chromatography and subsequent mass spectrometry. Metabolite fold changes in stimulated ciGEnC compared to untreated cells were calculated. RESULTS: 320 metabolites were identified and investigated. In response to TNFα + Stx2a, there was a predominant increase in intracellular free fatty acids and amino acids. Furthermore, lipid- and protein derived pro-inflammatory mediators, oxidative stress and an augmented intracellular energy turnover were increased in ciGEnC. Levels of most biochemicals related to carbohydrate metabolism remained unchanged. CONCLUSION: Stimulation of ciGEnC with TNFα + Stx2a is associated with profound metabolic changes indicative of increased inflammation, oxidative stress and energy turnover.
- Klíčová slova
- Conditionally immortalized glomerular endothelial cells, Hemolytic uremic syndrome, Metabolomics, Shiga toxin,
- MeSH
- endoteliální buňky cytologie účinky léků metabolismus MeSH
- glomerulus cytologie MeSH
- kultivované buňky MeSH
- lidé MeSH
- lipopolysacharidy MeSH
- metabolomika * MeSH
- multivariační analýza MeSH
- počet buněk MeSH
- shiga toxin 2 metabolismus farmakologie MeSH
- viabilita buněk účinky léků MeSH
- zánět chemicky indukované metabolismus patologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- lipopolysacharidy MeSH
- shiga toxin 2 MeSH
We found previously that white adipose tissue (WAT) hyperplasia in obese mice was limited by dietary omega-3 polyunsaturated fatty acids (omega-3 PUFA). Here we aimed to characterize the underlying mechanism. C57BL/6N mice were fed a high-fat diet supplemented or not with omega-3 PUFA for one week or eight weeks; mice fed a standard chow diet were also used. In epididymal WAT (eWAT), DNA content was quantified, immunohistochemical analysis was used to reveal the size of adipocytes and macrophage content, and lipidomic analysis and a gene expression screen were performed to assess inflammatory status. The stromal-vascular fraction of eWAT, which contained most of the eWAT cells, except for adipocytes, was characterized using flow cytometry. Omega-3 PUFA supplementation limited the high-fat diet-induced increase in eWAT weight, cell number (DNA content), inflammation, and adipocyte growth. eWAT hyperplasia was compromised due to the limited increase in the number of preadipocytes and a decrease in the number of endothelial cells. The number of leukocytes and macrophages was unaffected, but a shift in macrophage polarization towards a less inflammatory phenotype was observed. Our results document that the counteraction of eWAT hyperplasia by omega-3 PUFA in dietary-obese mice reflects an effect on the number of adipose lineage and endothelial cells.
- Klíčová slova
- adipocyte, cellularity, fat, nutrition, obesity, proliferation, white adipose tissue,
- MeSH
- bílá tuková tkáň účinky léků MeSH
- dieta s vysokým obsahem tuků MeSH
- endoteliální buňky účinky léků MeSH
- kyseliny mastné omega-3 aplikace a dávkování MeSH
- makrofágy účinky léků patologie MeSH
- myši inbrední C57BL MeSH
- myši MeSH
- proliferace buněk účinky léků MeSH
- tukové buňky cytologie účinky léků MeSH
- zánět patologie MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- kyseliny mastné omega-3 MeSH
Evaluating nanoparticle (NP) toxicity in human cell systems is a fundamental requirement for future NP biomedical applications. In this study, we have designed a screening assay for assessing different types of cell death induced by NPs in human umbilical vein endothelial cell (HUVEC) culture. This assay consists of WST-8, LDH and Hoechst 33342 staining, all performed in one well, which enables an evaluation of cell viability, necrosis and apoptosis, respectively, in the same cell sample. The 96-well format and automated processing of fluorescent images enhances the assay rapidity and reproducibility. After testing the assay functionality with agents that induced different types of cell death, we investigated the endothelial toxicity of superparamagnetic iron oxide nanoparticles (SPIONs, 8 nm), silica nanoparticles (SiNPs, 7-14 nm) and carboxylated multiwall carbon nanotubes (CNTCOOHs, 60 nm). Our results indicated that all the tested NP types induced decreases in cell viability after 24 hours at a concentration of 100 μg/ml. SPIONs caused the lowest toxicity in HUVECs. By contrast, SiNPs induced pronounced necrosis and apoptosis. A time course experiment showed the gradual toxic effect of all the tested NPs. CNTCOOHs inhibited tetrazolium derivatives at 100 μg/ml, causing false negative results from the WST-8 and LDH assay. In summary, our data demonstrate that the presented "three-in-one" screening assay is capable of evaluating NP toxicity effectively and reliably. Due to its simultaneous utilization of two different methods to assess cell viability, this assay is also capable of revealing, if NPs interfere with tetrazolium salts.
- MeSH
- apoptóza účinky léků MeSH
- biotest MeSH
- endoteliální buňky pupečníkové žíly (lidské) MeSH
- endoteliální buňky účinky léků MeSH
- lidé MeSH
- nanočástice aplikace a dávkování MeSH
- preklinické hodnocení léčiv metody MeSH
- velikost částic MeSH
- viabilita buněk účinky léků MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Objective- The leukocyte heme-enzyme MPO (myeloperoxidase) exerts proinflammatory effects on the vascular system primarily linked to its catalytic properties. Recent studies have shown that MPO, depending on its cationic charge, mediates neutrophil recruitment and activation. Here, we further investigated MPO's extracatalytic properties and its effect on endothelial glycocalyx (EG) integrity. Approach and Results- In vivo staining of murine cremaster muscle vessels with Alcian Blue 8GX provided evidence of an MPO-dependent decrease in anionic charge of the EG. MPO binding to the glycocalyx was further characterized using Chinese hamster ovary cells and its glycosaminoglycan mutants-pgsA-745 (mutant Chinese hamster ovary cells lacking heparan sulfate and chondroitin sulfate glycosaminoglycan) and pgsD-677 (mutant Chinese hamster ovary cells lacking heparan sulfate glycosaminoglycan), which revealed heparan sulfate as the main mediator of MPO binding. Further, EG integrity was assessed in terms of thickness using intravital microscopy of murine cremaster muscle. A significant reduction in EG thickness was observed on infusion of catalytically active MPO, as well as mutant inactive MPO and cationic polymer polylysine. Similar effects were also observed in wild-type mice after a local inflammatory stimulus but not in MPO-knockout mice. The reduction in EG thickness was reversed after removal of vessel-bound MPO, suggesting a possible physical collapse of the EG. Last, experiments with in vivo neutrophil depletion revealed that MPO also induced neutrophil-mediated shedding of the EG core protein, Sdc1 (syndecan-1). Conclusions- These findings provide evidence that MPO, via ionic interaction with heparan sulfate side chains, can cause neutrophil-dependent Sdc1 shedding and collapse of the EG structure.
- Klíčová slova
- glycocalyx, inflammation, intravital microscopy, peroxidase,
- MeSH
- aktivace neutrofilů MeSH
- břišní svaly krevní zásobení MeSH
- CHO buňky MeSH
- Cricetulus MeSH
- endoteliální buňky pupečníkové žíly (lidské) účinky léků metabolismus patologie MeSH
- endoteliální buňky účinky léků metabolismus patologie MeSH
- glykokalyx účinky léků metabolismus patologie MeSH
- heparansulfát proteoglykany metabolismus MeSH
- kationty MeSH
- lidé MeSH
- myši inbrední C57BL MeSH
- myši knockoutované MeSH
- neutrofily účinky léků metabolismus MeSH
- peroxidasa nedostatek genetika metabolismus farmakologie MeSH
- syndekan-1 metabolismus MeSH
- vazba proteinů MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- heparansulfát proteoglykany MeSH
- kationty MeSH
- MPO protein, human MeSH Prohlížeč
- peroxidasa MeSH
- SDC1 protein, human MeSH Prohlížeč
- syndekan-1 MeSH
Incomplete endothelialization of intracoronary stents has been associated with stent thrombosis and recurrent symptoms, whereas prolonged use of dual antiplatelet therapy increases bleeding-related adverse events. Facilitated endothelialization has the potential to improve clinical outcomes in patients who are unable to tolerate dual antiplatelet therapy. The objective of this study was to demonstrate the feasibility of magnetic cell capture to rapidly endothelialize intracoronary stents in a large animal model. A novel stent was developed from a magnetizable duplex stainless steel (2205 SS). Polylactic-co-glycolic acid and magnetite (Fe3O4) were used to synthesize biodegradable superparamagnetic iron oxide nanoparticles, and these were used to label autologous blood outgrowth endothelial cells. Magnetic 2205 SS and nonmagnetic 316L SS control stents were implanted in the coronary arteries of pigs (n = 11), followed by intracoronary delivery of magnetically labeled cells to 2205 SS stents. In this study, we show extensive endothelialization of magnetic 2205 SS stents (median 98.4% cell coverage) within 3 days, whereas the control 316L SS stents exhibited significantly less coverage (median 48.9% cell coverage, p < 0.0001). This demonstrates the ability of intracoronary delivery of magnetic nanoparticle labeled autologous endothelial cells to improve endothelialization of magnetized coronary stents within 3 days of implantation.
- Klíčová slova
- cell-targeting, endothelialization, magnetic stent, nanotechnology,
- MeSH
- endoteliální buňky cytologie účinky léků ultrastruktura MeSH
- fenotyp MeSH
- kovy chemie MeSH
- nanočástice chemie ultrastruktura MeSH
- nerezavějící ocel farmakologie MeSH
- prasata MeSH
- stenty * MeSH
- zvířata MeSH
- Check Tag
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
- Názvy látek
- kovy MeSH
- nerezavějící ocel MeSH
