The influence of redox modulation on hypoxic endothelial cell metabolic and proteomic profiles through a small thiol-based compound tuning glutathione and thioredoxin systems

Reduction in oxygen levels is a key feature in the physiology of the bone marrow (BM) niche where hematopoiesis occurs. The BM niche is a highly vascularized tissue and endothelial cells (ECs) support and regulate blood cell formation from hematopoietic stem cells (HSCs). While in vivo studies are limited, ECs when cultured in vitro at low O-2 (<5%), fail to support functional HSC maintenance due to oxidative environment. Therefore, changes in EC redox status induced by antioxidant molecules may lead to alterations in the cellular response to hypoxia likely favoring HSC self-renewal. To evaluate the impact of redox regulation, HUVEC, exposed for 1, 6, and 24 h to 3% O-2 were treated with N-(N-acetyl-l-cysteinyl)-S-acetylcysteamine (I-152). Metabolomic analyses revealed that I-152 increased glutathione levels and influenced the metabolic profiles interconnected with the glutathione system and the redox couples NAD(P)+/NAD(P)H. mRNA analysis showed a lowered gene expression of HIF-1a and VEGF following I-152 treatment whereas TRX1 and 2 were stimulated. Accordingly, the proteomic study revealed the redox-dependent upregulation of thioredoxin and peroxiredoxins that, together with the glutathione system, are the main regulators of intracellular ROS. Indeed, a time-dependent ROS production under hypoxia and a quenching effect of the molecule were evidenced. At the secretome level, the molecule downregulated IL-6, MCP-1, and PDGF-bb. These results suggest that redox modulation by I-152 reduces oxidative stress and ROS level in hypoxic ECs and may be a strategy to fine-tune the environment of an in vitro BM niche able to support functional HSC maintenance.

Automated summary

Reduction in oxygen levels is a key feature in the physiology of the bone marrow (BM) niche where hematopoiesis occurs. Antioxidant molecules can modulate the redox status of endothelial cells (ECs) in a hypoxic environment, promoting hematopoietic stem cell (HSC) maintenance. In this study, HUVEC cells exposed to low oxygen were treated with I-152, which increased glutathione levels and influenced the metabolic profiles interconnected with the glutathione system. Gene and protein expression analyses showed modulation of redox-related genes and upregulation of antioxidants. I-152 also reduced oxidative stress and downregulated certain cytokines in hypoxic ECs, potentially creating an in vitro BM niche conducive to functional HSC maintenance.