Adenosine Prevents TNFα-Induced Decrease in Endothelial Mitochondrial Mass via Activation of eNOS-PGC-1α Regulatory Axis

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.

Suppression of endothelial PGC-1α is associated with hypoxia-induced endothelial dysfunction and provides a new therapeutic target in pulmonary arterial hypertension

“…NO could upregulate PGC-1␣ (8) and the eNOS-PGC-1␣ regulatory pathway, which preserves endothelial mitochondrial function. PGC-1␣-mediated protection plays a role in preconditioning strategies; this protection is multifactorial and not limited to mitochondrial biogenesis (28).…”

Endothelial Cells Harbouring the JAK2V617F Mutation Display Pro-Adherent and Pro-Thrombotic Features

“…Nitric oxide (NO) production was detected by incubating ELCs grown to confluence and depleted overnight from growth factors with 10 μM 4-amino-5-methylamino-2',7'difluorofluorescein diacetate (DAF-FM DA) (sc-205940, Santa Cruz Biotechnology) for 30 minutes, following a protocol adapted from the literature. 15,16 Cells were then washed, and NO synthesis, estimated by DAF-FM DA green fluorescence, was monitored during the following 15 minutes by image acquisition using the AxioObserver Z1 microscope (objective 10Â) and AxioVision 4 software (Carl Zeiss). Intensities of fluorescence were normalized to the cell surface area by using the Icy software.…”

Protective Effects of Methotrexate against Proatherosclerotic Cytokines: A Review of the Evidence

“…The pretreatment of endothelial cells with TNF- α did not affect the methotrexate-mediated upregulation of manganese superoxide dismutase and haem oxygenase-1 mRNA [ 89 ]. Methotrexate (10 −8 M to 10 −6 M) and/or adenosine treatment has also been shown to prevent the TNF- α -induced (a) expression of ICAM-1 and VCAM-1 in human umbilical vein endothelial cells (HUVEC) [ 90 ] and (b) reduction of mitochondrial mass, membrane potential, and intracellular ATP concentrations, in endothelial cells through increased activity of eNOS [ 91 ]. Furthermore, adenosine has been shown to reduce the circulating concentrations of TNF- α and IL-6 in a mice model of sepsis ( Table 2 ) [ 92 ].…”