Cold induces reactive oxygen species production and activation of the NF-kappa B response in endothelial cells and inflammation in vivo.

BACKGROUND: Organs intended for transplantation are generally stored in the cold for better preservation of their function. However, following transplantation and reperfusion, the microvasculature of transplanted organs often proves to be activated. Extensive leukocyte adhesion and microthrombus formation contribute to failure of the transplanted organ. OBJECTIVES: In this study we analyzed cold-induced changes to the activation status of cultured endothelial cells, possibly contributing to organ failure. METHODS: We exposed human umbilical vein endothelial cells (HUVECs) to temperatures below 37 °C (mostly to 8 °C) for 30 min and upon rewarming to 37 °C kept incubating them for up to 24 h. We also in vivo locally exposed mice to cold. RESULTS: The exposure to low temperatures induced, in HUVECs, expression of the prothrombotic factors plasminogen activator inhibitor-1 (PAI-1) and tissue factor (TF) and of the inflammatory adhesion molecules, E-selectin, intracellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). Furthermore, upon rewarming for 30 min, we detected activation of the inflammatory NF-κB pathway, as measured by transient NF-κB translocation to the nucleus and IκBα degradation. Using butylated hydroxytoluene (BHT), a scavenger of reactive oxygen species (ROS), we further demonstrated that cold-induced NF-κB activation depends on ROS production. Local exposure to cold also, in vivo, induced ROS production and ICAM-1 expression and resulted in leukocyte infiltration. CONCLUSIONS: Our results point to a causative link between ROS production and NF-κB activation, suppression of which had been shown to be beneficial during hypothermic storage and subsequent rewarming of organs for transplantation.

A carbon-source-responsive element is required for regulation of the hypoxic ADP/ATP carrier (AAC3) isoform in Saccharomyces cerevisiae.

The mitochondrial ADP/ATP carrier in Saccharomyces cerevisiae is encoded by three genes that are differentially expressed under different physiological conditions. We investigated the transcriptional control of AAC3, an oxygen-repressed isoform. By deletion analysis, DNA electrophoretic mobility-shift assays, DNase I footprinting and site-directed mutagenesis, we have identified a promoter region (upstream repressing sequence 1, URS(1)) involved in a carbon-source-dependent repression of AAC3. It is different from the previously characterized oxygen-dependent ROX1 (regulation by oxygen 1) repressor-binding region (URS(2)). The complex character of URS(1) includes the presence of two different cis-acting sequences: (i) a RAP1 (repressor activator protein 1)-binding site that is capable of binding the RAP1 protein in vitro and (ii) two putative ethanol-repression sequences, the modification of which derepresses the AAC3 gene. These findings demonstrate that the hypoxic AAC3 gene is regulated by two upstream repressor sites; one controlled by oxygen and haem, the other by the carbon source. Both sites function to completely switch off the expression of the AAC3 isoform when ATP is made by oxidative phosphorylation, and they modulate AAC3 expression when import of glycolytic ATP into mitochondria is required.

Enhanced mitochondrial biogenesis is associated with increased expression of the mitochondrial ATP-dependent Lon protease.

Rats bearing the Zajdela hepatoma tumor and T3-treated hypothyroid rats were used to study the role of protein degradation in the process of mitochondrial biogenesis. It was shown that the activity, protein and mRNA levels of the ATP-dependent Lon protease increased in rapidly growing Zajdela hepatoma cells. The increase in the rate of mitochondrial biogenesis by thyroid hormone was similarly accompanied by enhanced expression of the Lon protease. The results imply that mitochondrial biogenesis in mammalian cells is, at least partially, regulated by the matrix Lon protease.