Abstract
We tested whether adenosine, a cytoprotective mediator and trigger of preconditioning, could protect endothelial cells from inflammation-induced deficits in mitochondrial biogenesis and function. We examined this question using human microvascular endothelial cells exposed to TNFα. TNFα produced time and dose-dependent decreases in mitochondrial membrane potential, cellular ATP levels, and mitochondrial mass, preceding an increase in apoptosis. These effects were prevented by co-incubation with adenosine, a nitric oxide (NO) donor, a guanylate cyclase (GC) activator, or a cell-permeant cyclic GMP (cGMP) analog. The effects of adenosine were blocked by a nitric oxide synthase inhibitor, a soluble guanylate cyclase inhibitor, a morpholino antisense oligonucleotide to endothelial nitric oxide synthase (eNOS), or siRNA knockdown of the transcriptional coactivator, PGC-1α. Incubation with exogenous NO, a GC activator, or a cGMP analog reversed the effect of eNOS knockdown, while the effect of NO was blocked by inhibition of GC. The protective effects of NO and cGMP analog were prevented by siRNA to PGC-1α. TNFα also decreased expression of eNOS, cellular NO levels, and PGC-1α expression, which were reversed by adenosine. Exogenous NO, but not adenosine, rescued expression of PGC-1α in cells in which eNOS expression was knocked down by eNOS antisense treatment. Thus, TNFα elicits decreases in endothelial mitochondrial function and mass, and an increase in apoptosis. These effects were reversed by adenosine, an effect mediated by eNOS-synthesized NO, acting via soluble guanylate cyclase/cGMP to activate a mitochondrial biogenesis regulatory program under the control of PGC-1α. These results support the existence of an adenosine-triggered, mito-and cytoprotective mechanism dependent upon an eNOS-PGC-1α regulatory pathway, which acts to preserve endothelial mitochondrial function and mass during inflammatory challenge.
Highlights
The process of mitochondrial biogenesis–the coordinated orchestration of nuclear and mitochondrial gene expression, mitochondrial protein import, and structural dynamics, so as to optimize cellular mitochondrial function–has recently been proposed as a potentially useful therapeutic target in the protective effects of ischemic preconditioning (IPC) [1]
In experiments testing the effects of adenosine (10 mM), (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl) amino] diazen-1-ium-1, 2-diolate, N5-(1-iminoethyl)-L-ornithine, dihydrochloride (LNIO,100 mM), 1H-(1,2,4)-oxadiazolo-[4,3-alpha]-quinozalin-1one (ODQ, 30 mM), 3-(59-hydroxymethyl-29-furyl)-1-benzyl indazole (YC-1, 100 mM), and 8-bromo-cyclic GMP (8-Br-cGMP, 500 mM), each was added to cells immediately prior to TNFa both at the start of the experiment, and again at 24 h. endothelial nitric oxide synthase (eNOS) antisense and PGC-1a siRNA experiments: The role of endogenous eNOS in mediating the effect of adenosine on TNFa-induced decrease in mitochondrial mass was tested using cells that had been transfected with a morpholino antisense oligomer construct (Gene Tools, Philomath, OR) to eNOS
In order to distinguish between these two possibilities, we examined the ability of adenosine and the nitric oxide (NO) donor, detaNO to reverse TNFa’s effect on mitotracker green (MTG) fluorescence under conditions where expression of PGC-1a had been knocked down using an siRNA
Summary
The process of mitochondrial biogenesis–the coordinated orchestration of nuclear and mitochondrial gene expression, mitochondrial protein import, and structural dynamics, so as to optimize cellular mitochondrial function–has recently been proposed as a potentially useful therapeutic target in the protective effects of ischemic preconditioning (IPC) [1]. It is not known to what extent mitochondrial biogenesis in the endothelium might play in these processes, it is reasonable to propose such a role, by virtue of this organelle’s recognized function as a platform for coordination of redox-dependent cell signaling and cell death [8,17,18,19,20]. Results from studies in other cell/tissue types are conflicting It has been shown in several cell types that improvements in mitochondrial reserve capacity and/or function might be explained by increases in mitochondrial mass [22,23,24]. Other studies in heart and skeletal muscle have reported a dissociation between mitochondrial mass and function [15,16,25] Examination of this issue in endothelial cells has not been reported
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