Nitric oxide protects the heart from ischemia-induced apoptosis and mitochondrial damage via protein kinase G mediated blockage of permeability transition and cytochrome c release.

BACKGROUND: Heart ischemia can rapidly induce apoptosis and mitochondrial dysfunction via mitochondrial permeability transition-induced cytochrome c release. We tested whether nitric oxide (NO) can block this damage in isolated rat heart, and, if so, by what mechanisms. METHODS: Hearts were perfused with 50 microM DETA/NO (NO donor), then subjected to 30 min stop-flow ischemia or ischemia/reperfusion. Isolated heart mitochondria were used to measure the rate of mitochondrial oxygen consumption and membrane potential using oxygen and tetraphenylphosphonium-selective electrodes. Mitochondrial and cytosolic cytochrome c levels were measured spectrophotometrically and by ELISA. The calcium retention capacity of isolated mitochondria was measured using the fluorescent dye Calcium Green-5N. Apoptosis and necrosis were evaluated by measuring the activity of caspase-3 in cytosolic extracts and the activity of lactate dehydrogenase in perfusate, respectively. RESULTS: 30 min ischemia caused release of mitochondrial cytochrome c to the cytoplasm, inhibition of the mitochondrial respiratory chain, and stimulation of mitochondrial proton permeability. 3 min perfusion with 50 microM DETA/NO of hearts prior to ischemia decreased this mitochondrial damage. The DETA/NO-induced blockage of mitochondrial cytochrome c release was reversed by a protein kinase G (PKG) inhibitor KT5823, or soluble guanylate cyclase inhibitor ODQ or protein kinase C inhibitors (Ro 32-0432 and Ro 31-8220). Ischemia also stimulated caspase-3-like activity, and this was substantially reduced by pre-perfusion with DETA/NO. Reperfusion after 30 min of ischemia caused no further caspase activation, but was accompanied by necrosis, which was completely prevented by DETA/NO, and this protection was blocked by the PKG inhibitor. Incubation of isolated heart mitochondria with activated PKG blocked calcium-induced mitochondrial permeability transition and cytochrome c release. Perfusion of non-ischemic heart with DETA/NO also made the subsequently isolated mitochondria resistant to calcium-induced permeabilisation, and this protection was blocked by the PKG inhibitor. CONCLUSION: The results indicate that NO rapidly protects the ischemic heart from apoptosis and mitochondrial dysfunction via PKG-mediated blockage of mitochondrial permeability transition and cytochrome c release.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

Efficacy of proanthocyanidins from Pelargonium sidoides root extract in reducing P. gingivalis viability while preserving oral commensal S. salivarius

Funding Information: Funding: This research is a part of the project PELARGODONT (“Engineering and functionalization of delivery system with Pelargonium sidoides biologically active substance on inflamed periodontal surface area”) funded by a grant (No. S-M-ERA.NET-17-2) from the Research Council of Lithuania, the State Education Development Agency of Latvia, and Italian Ministry of Education, University and Research. Publisher Copyright: © 2018 by the authors.Bacterial resistance to antibiotics and the disruption of beneficial microbiota are key problems in contemporary medicine and make the search for new, more efficient infection treatment strategies among the most important tasks in medicine. Multicomponent plant-derived preparations with mild antibacterial activity created by many simultaneous mechanisms together with anti-inflammatory, innate immune and regenerative capacity-stimulating properties are good candidates for this therapy, and proanthocyanidins are among the most promising compounds of this sort. In this study, we have isolated proanthocyanidins from Pelargonium sidoides DC root extract and characterized and compared the composition, antioxidant properties and antibacterial activity of the proanthocyanidin fraction with those of the whole extract. The results revealed that proanthocyanidins had significantly stronger antioxidant capacity compared to the root extract and exhibited a unique antibacterial action profile that selectively targets Gram-negative keystone periodontal and peri-implant pathogenic strains, such as Porphyromonas gingivalis, while preserving the viability of beneficial oral commensal Streptococcus salivarius. The finding suggests that proanthocyanidins from Pelargonium sidoides root extract are good candidates for the prolonged and harmless treatment of infectious diseases.publishersversionPeer reviewe

Comparison of aqueous, polyethylene glycol-aqueous and ethanolic propolis extracts: antioxidant and mitochondria modulating properties

Abstract Background Propolis is multicomponent substance collected by honeybees from various plants. It is known for numerous biological effects and is commonly used as ethanolic extract because most of active substances of propolis are ethanol-soluble. However, water-based propolis extracts could be applied more safely, as this solvent is more biocompatible. On the other hand, water extracts has significantly smaller range and quantity of active compounds. The extraction power of water could be enhanced by adding co-solvent which increases both solubility and penetration of propolis compounds. However, variation of solvents results in different composition of active substances that might have distinct effects. The majority of biological effects of propolis are attributed to the antioxidant properties of its active compounds. Antioxidant effect might be a result of either direct scavenging of ROS or modulation of ROS producing organelle activity. Therefore, the aim of this study was to investigate and compare chemical composition, antioxidant properties and effects on mitochondrial respiration of aqueous (AqEP), polyethylene glycol-aqueous (Pg-AqEP) and ethanolic (EEP) propolis extracts. Methods Chemical composition of propolis extracts was determined using HPLC and Folin-Ciocalteu method. Ability to neutralize H2O2 and intracellular ROS concentration in C6 glioma cells were determined fluorometrically by using 10-acetyl-3,7-dihydroxyphenoxazine and 2′,7′-dichlorofluorescein diacetate, respectively. Mitochondrial superoxide generation was assessed under fluorescent microscope by using MitoSOX Red. Oxygen uptake rates of mitochondria were recorded by high-resolution respirometer Oxygraph-2 k. Results Our data revealed that phenolic acids and aldehydes make up 40–42% of all extracted and identified compounds in AqEP and Pg-AqEP and only 16% in EEP. All preparations revealed similar antioxidant activity in cell culture medium but Pg-AqEP and EEP demonstrated better mitochondrial superoxide and total intracellular ROS decreasing properties. At higher concentrations, AqEP and EEP inhibited mitochondrial respiration, but Pg-AqEP had concentration-dependent mitochondria-uncoupling effect. Conclusions Aqueous and non-aqueous propolis extracts differ by composition, but all of them possess antioxidant properties and neutralize H2O2 in solution at similar efficiency. However, both Pg-AqEP and EEP were more effective in decreasing intracellular and intramitochondrial ROS compared to AqEP. At higher concentrations, these preparations affect mitochondrial functions and change energy production in C6 cells

Investigation of Cancer Cell Migration and Proliferation on Synthetic Extracellular Matrix Peptide Hydrogels

Chemical and mechanical properties of a tumor microenvironment are essential players in cancer progression, and it is important to precisely control the extracellular conditions while designing cancerin vitromodels. The study investigates synthetic hydrogel matrices from multi-arm polyethylene glycol (PEG) functionalized with collagen-like peptide (CLP) CG(PKG)(4)(POG)(4)(DOG)(4)alone and conjugated with either cell adhesion peptide RGD (mimicking fibronectin) or IKVAV (mimicking laminin). Human glioblastoma HROG36, rat C6 glioma cells, and A375 human melanoma cells were grown on the hydrogels and monitored for migration, proliferation, projected cell area, cell shape index, size and number, distribution of focal contacts in individual cells, and focal adhesion number. PEG-CLP-RGD induced migration of both glioma cell lines and also stimulated proliferation (assessed as metabolic activity) of HROG36 cells. Migration of C6 cells were also stimulated by PEG-CLP-IKVAV. These responses strongly correlated with the changes in adhesion and morphology parameters of individual cells - projected cell area, cell shape index, and focal contact number. Melanoma A375 cell proliferation was increased by PEG-CLP-RGD, and this was accompanied by a decrease in cell shape index. However, neither RGD nor IKVAV conjugated to PEG-CLP stimulated migratory capacity of A375 cells. Taken together, the study presents synthetic scaffolds with extracellular matrix (ECM)-mimicking peptides that allow for the exploration of the effect of ECM signaling to cancer cells.Funding Agencies|Research Council of Lithuania [01.2.2-LMT-K-718-01-0036]</p

Cerebellar Cells Self-Assemble into Functional Organoids on Synthetic, Chemically Crosslinked ECM-Mimicking Peptide Hydrogels

Hydrogel-supported neural cell cultures are more in vivo-relevant compared to monolayers formed on glass or plastic substrates. However, there is a lack of synthetic microenvironment available for obtaining standardized and easily reproducible cultures characterized by tissue-mimicking cell composition, cell-cell interactions, and functional networks. Synthetic peptides representing the biological properties of the extracellular matrix (ECM) proteins have been reported to promote the adhesion-driven differentiation and functional maturation of neural cells. Thus, such peptides can serve as building blocks for engineering a standardized, all-synthetic environment. In this study, we have compared the effect of two chemically crosslinked hydrogel compositions on primary cerebellar cells: collagen-like peptide (CLP), and CLP with an integrin-binding motif arginine-glycine-aspartate (CLP-RGD), both conjugated to polyethylene glycol molecular templates (PEG-CLP and PEG-CLP-RGD, respectively) and fabricated as self-supporting membranes. Both compositions promoted a spontaneous organization of primary cerebellar cells into tissue-like clusters with fast-rising Ca2+ signals in soma, reflecting action potential generation. Notably, neurons on PEG-CLP-RGD had more neurites and better synaptic efficiency compared to PEG-CLP. For comparison, poly-L-lysine-coated glass and plastic surfaces did not induce formation of such spontaneously active networks. Additionally, contrary to the hydrogel membranes, glass substrates functionalized with PEG-CLP and PEG-CLP-RGD did not sufficiently support cell attachment and, subsequently, did not promote functional cluster formation. These results indicate that not only chemical composition but also the hydrogel structure and viscoelasticity are essential for bioactive signaling. The synthetic strategy based on ECM-mimicking, multifunctional blocks in registry with chemical crosslinking for obtaining tissue-like mechanical properties is promising for the development of fast and well standardized functional in vitro neural models and new regenerative therapies