संवहनी Ca के विज़ुअलाइज़ेशन 2 + संकेतन Paracrine व्युत्पन्न ROS द्वारा ट्रिगर
Summary
Paracrine व्युत्पन्न endothelial सीए 2 + संकेत के ROS प्रेरण visualizing में अंतर्दृष्टि लाभ के लिए एक कुशल विधि वर्णित है. इस विधि paracrine व्युत्पन्न ROS मापने संवहनी endothelial कोशिकाओं में एक सह संस्कृति मॉडल में सीए 2 + जुटाना ट्रिगर का लाभ लेता है.
Abstract
Oxidative stress has been implicated in a number of pathologic conditions including ischemia/reperfusion damage and sepsis. The concept of oxidative stress refers to the aberrant formation of ROS (reactive oxygen species), which include O2•-, H2O2, and hydroxyl radicals. Reactive oxygen species influences a multitude of cellular processes including signal transduction, cell proliferation and cell death1-6. ROS have the potential to damage vascular and organ cells directly, and can initiate secondary chemical reactions and genetic alterations that ultimately result in an amplification of the initial ROS-mediated tissue damage. A key component of the amplification cascade that exacerbates irreversible tissue damage is the recruitment and activation of circulating inflammatory cells. During inflammation, inflammatory cells produce cytokines such as tumor necrosis factor-α (TNFα) and IL-1 that activate endothelial cells (EC) and epithelial cells and further augment the inflammatory response7. Vascular endothelial dysfunction is an established feature of acute inflammation. Macrophages contribute to endothelial dysfunction during inflammation by mechanisms that remain unclear. Activation of macrophages results in the extracellular release of O2•- and various pro-inflammatory cytokines, which triggers pathologic signaling in adjacent cells8. NADPH oxidases are the major and primary source of ROS in most of the cell types. Recently, it is shown by us and others9,10 that ROS produced by NADPH oxidases induce the mitochondrial ROS production during many pathophysiological conditions. Hence measuring the mitochondrial ROS production is equally important in addition to measuring cytosolic ROS. Macrophages produce ROS by the flavoprotein enzyme NADPH oxidase which plays a primary role in inflammation. Once activated, phagocytic NADPH oxidase produces copious amounts of O2•- that are important in the host defense mechanism11,12. Although paracrine-derived O2•- plays an important role in the pathogenesis of vascular diseases, visualization of paracrine ROS-induced intracellular signaling including Ca2+ mobilization is still hypothesis. We have developed a model in which activated macrophages are used as a source of O2•- to transduce a signal to adjacent endothelial cells. Using this model we demonstrate that macrophage-derived O2•- lead to calcium signaling in adjacent endothelial cells.
Protocol
Discussion
विधि यहाँ वर्णित कोशिकाओं रहने वाले या तो ऑक्सीकरण संवेदनशील रंजक या प्लाज्मिड सेंसर का उपयोग करने में प्रतिक्रियाशील ऑक्सीजन प्रजातियों के तेजी से और मात्रात्मक माप की अनुमति देता है. TLRs के agonists (टोल ?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
यह काम राष्ट्रीय संस्थानों स्वास्थ्य अनुदान के एम.एम. (HL086699 R01, HL086699 01A2S1, 1S10RR027327-01) द्वारा समर्थित किया गया. हमारे लेख आंशिक रूप से कार्ल Zeiss MicroImaging LLC द्वारा समर्थित है.
Materials
| Reagent | Company | Catalogue number |
| Attofluor cell chamber | Invitrogen | A7816 |
| Antimycin A | Sigma Aldrich | A8674 |
| DMEM low glucose medium | Invitrogen | 10567-014 |
| Endothelial growth factor supplement (ECGS) | Upstate | 02-102 |
| Fetal Bovine Serum | Invitrogen | 12662011 |
| G418 | Invitrogen | 10131-027 |
| Gelatin | Sigma Aldrich | G1393 |
| H2DCFDA | Invitrogen | D-399 |
| LPS | Sigma Aldrich | L4516 |
| LTA | Sigma Aldrich | L2515 |
| MitoSOX Red | Invitrogen | M36008 |
| Opti-MEM I Reduced Serum Medium | Invitrogen | 51985091 |
| Pen/Strep (10x) | Invitrogen | 15140163 |
| pHyPer-cyto | Evrogen | FP941 |
| pHyPer-dMito | Evrogen | FP942 |
| Poly(I:C) | Sigma Aldrich | P0913 |
| Prism software 5.0 | GraphPad Software, Inc. | |
| SigmaPlot 11.0 | Systat software, Inc. | |
| Trypsin-EDTA (10x) | Invitrogen | 15400054 |
| T-25 Flasks | Corning | 430639 |
| T-75 Flasks | BD Falcon | 353136 |
| 96-well TC micro well plate | BD Falcon | 353072 |
| Zen 2009 software | Carl Zeiss 510 Meta confocal microscopy |
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