Visualization of Vascular Ca2+ Signaling Triggered by Paracrine Derived ROS

Summary

An efficient method to gain insights into visualizing the paracrine-derived ROS induction of endothelial Ca2+ signaling is described. This method takes advantage of measuring paracrine derived ROS triggered Ca2+ mobilization in vascular endothelial cells in a co-culture model.

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 death^1-6. 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 others^9,10 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 mechanism^11,12. 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

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

1. Grow J774.1 mouse monocyte-derived macrophages (10⁶ cells/ml) on glass bottom 35-mm dishes (Harvard Apparatus) for 48 h.
2. Challenge cells with TLR agonists (2 μg/ml, Lipo-teichoic acid-TLR2 agonist; 10μg/ml, Poly (I:C)-TLR3 agonist; 1 μg/ml LPS-TLR4 agonist) for 6 h at 37°C.
   Note: Macrophages treated under similar conditions were used for co-culture model Ca²⁺ mobilization.
3. Add the oxid....

Discussion

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 m.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Attofluor cell chamber	Invitrogen	A7816
Antimycin A	Sigma-Aldrich	A8674
DMEM low glucose medium	Invitrogen	10567-014
Endothelial growth factor supplement (ECGS)	Upstate, Millipore	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 Biosciences	353136
96-well TC micro well plate	BD Biosciences	353072
Zen 2009 software	Carl Zeiss, Inc.