1Department of Biochemistry, Temple University, 2Department of Anesthesiology and Pain Medicine, University of Washington
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Mallilankaraman, K., Gandhirajan, R. K., Hawkins, B. J., Madesh, M. Visualization of Vascular Ca2+ Signaling Triggered by Paracrine Derived ROS. J. Vis. Exp. (58), e3511, doi:10.3791/3511 (2011).
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.
Reactive oxygen species can be measured in live cells using oxidation sensitive dyes (1 & 2) or using plasmid sensors (3 & 4) by confocal microscopy.
1. Visualization of cytosolic ROS in J774 cells
2. Visualization of mROS in J774 cells
3. Visualization of cytosolic ROS in stable HyPer-cyto MPMVECs
4. Visualization of mitochondrial ROS in stable HyPer-dMito MPMVECs
5. Co-culture model for visualization of paracrine-derived ROS triggered Ca2+ signaling in endothelial cells
For the visualization and measurement of [Ca2+]i changes we use a Ca2+ sensitive fluorescent dye fluo-4 AM (Invitrogen).
6. Data analysis using ImageJ software
Transfer the images obtained from the confocal system as a .lsm format file for data analysis. ZEN 2009 and ZEN 2010 software provided with LSM 510 Meta confocal microscope or LSM 710 multiphoton confocal microscope respectively saves files in .lsm format. We recommend using this native .lsm files for image analysis as original quality is retained. This file format can be directly opened in image analysis program Image J. The following steps provide an example quantitative analysis of single image. No additional plugins are required for this data analysis.
7. Representative Results
Figure 1 shows cytosolic and mitochondrial ROS production upon challenge with TLR ligands. Representative confocal images show increase of DCF (cytosolic) and MitoSOX Red (mitochondria) fluorescence after 6 hr of stimulation. Fluorescence changes were normalized and expressed as fold change.
Figure 2 shows the generation of stable HyPer-cyto and HyPer-dMito endothelial cells. Mouse endothelial cells were transfected with pHyPer-cyto or pHyPer-dMito plasmid constructs via electroporation. Cells stably expressing plasmids were selected with G418 for two weeks. After selection, diluted cells were seeded in 96 well plates. Individual colonies were isolated and imaged for homogeneous expression.
Figure 3 shows the measurement of endothelial cytosolic and mitochondrial H2O2 levels. Representative confocal images show increase of HyPer-cyto (cytosolic) and HyPer-dMito (mitochondria) fluorescence after 6 hr of stimulation. Fluorescence intensity changes were normalized and expressed as fold change.
Figure 4 shows the assessment of macrophage-derived ROS induced endothelial cytosolic Ca2+ mobilization. LPS-stimulated macrophages were added onto pulmonary microvascular endothelial cells (PMVEC) that had been previously loaded with the intracellular Ca2+ ([Ca2+]i) indicator dye Fluo-4. Application of wild type LPS-activated macrophages evoked an [Ca2+]i rise in PMVECs that was attenuated by knockout of functional NADPH oxidase (gp91phox-/-).
Figure 1. Visualization of macrophage generated cellular ROS and mitochondrial superoxide. J774.1 cells were challenged with TLR2, 3 and 4 ligands. (2 μg/ml, Lipoteichoic acid-TLR2; 10μg/ml, Poly (I:C)-TLR3; 1 μg/ml LPS-TLR4) for 6 h at 37°C. After treatment cells were loaded with (A) cellular ROS indicator (H2DCF-DA; green fluorescence) or (B) mitochondrial superoxide indicator (MitoSOX Red; red fluorescence). (C) Antimycin A was used as a positive control for mitochondrial ROS production. Fluorescence intensity changes were assessed using confocal microscopy. (D) and (E) Quantitation of mean fluorescence change.
Figure 2. Schematic representation of generating stable expression of endothelial cell cytosolic and mitochondrial ROS indicators.
Figure 3. Visualization of endothelial cytosolic and mitochondrial H2O2 levels. MPMVECs stably expressing (A) HyPer-cyto or (B) HyPer-dMito were challenged with TLR2, 3 and 4 ligands (2 μg/ml, Lipoteichoic acid-TLR2; 10μg/ml, Poly (I:C)-TLR3; 1 μg/ml LPS-TLR4) for 6 h at 37°C. (C) Antimycin A was used as a positive control for mitochondrial ROS production. After treatment, fluorescence intensity changes were assessed using confocal microscopy. (D) and (E) Quantitation of mean fluorescence change.
Figure 4. Macrophage-derived ROS elicit impaired endothelial cell Ca2+ mobilization. (A) Schematic representation of endothelial cells/macrophage co-culture model study. (B) Freshly isolated murine macrophages from both WT and gp91phox null mice (The Jackson Laboratory) were activated by 1 μg/ml LPS for 6 h. Macrophages were labeled with cell-tracker Red (red) and added onto Fluo-4 loaded PMVECs (green) to assess paracrine O2•- signaling. (C) LPS-activated macrophages isolated from WT mice triggered large and rapid Ca2+ mobilization in PMVECs compared to macrophages from gp91phox null mice. Validation of macrophage activation has been demonstrated previously.
The method described here allows rapid and quantitative measurement of reactive oxygen species in living cells either using oxidation sensitive dyes or plasmid sensors. Agonists of TLRs (Toll-like receptors) are compounds that stimulate the cells through the TLRs present on the cell surface and trigger the downstream signaling pathways15. In our protocol, we used three different TLR agonists viz., Lipo-teichoic acid-TLR2 agonist; Poly (I:C)-TLR3 agonist; LPS-TLR4 agonist which are reported to induce ROS as a model system to demonstrate the ROS measurement. Measurement of ROS using oxidants sensitive dye is rapid and can be used to measure variety of ROS species. The use of plasmid based sensor HyPer selectively detects H2O2 levels. H2O2 is more stable oxidant and also the use of HyPer cyto and HyPer-dMito in a stable cell setting adds the advantage over photo-autooxidation of ROS sensitive fluorescent dyes. HyPer does not cause artifactual ROS generation and can be used for detection of fast changes of H2O2 concentration in different cell compartments under various physiological and pathological conditions. Cells stably expressing the HyPer sensors are used for clonal selection of population that offers more reliable and accurate measurements of H2O2 levels. Importantly, this method also describes the detection and quantitative measurement of Ca2+ mobilization in live cells triggered by paracrine derived ROS from the neighboring cells such as innate immune cells. This experimental procedure combined with confocal microscopy may be very useful to reveal important spatio-temporal changes in cell signal transduction events especially in instances where two different cell types are involved. Though, the representative experiments detailed in this paper are just one example of the type of interaction studied, this technique can be easily adapted to study cellular interactions between multiple live cell types.
No conflicts of interest declared.
This work was supported by the National Institutes of Health grant (R01 HL086699, HL086699-01A2S1, 1S10RR027327-01) to MM. Our article is partly supported by Carl Zeiss Microimaging LLC.
|Attofluor cell chamber||Invitrogen||A7816|
|DMEM low glucose medium||Invitrogen||10567-014|
|Endothelial growth factor supplement (ECGS)||Upstate, Millipore||02-102|
|Fetal Bovine Serum||Invitrogen||12662011|
|Opti-MEM I Reduced Serum Medium||Invitrogen||51985091|
|Prism software 5.0||GraphPad Software Inc.|
|SigmaPlot 11.0||Systat software, Inc.|
|T-75 Flasks||BD Biosciences||353136|
|96-well TC micro well plate||BD Biosciences||353072|
|Zen 2009 software||Carl Zeiss, Inc.|