51 - CSE derived sulfur metabolites regulates autophagy in ischemic endothelial cells

Abstract

Background Peripheral arterial disease (PAD) causes narrowing of blood vessels resulting in restriction of blood flow to lower limbs. Recently, autophagy has progressed as an emerging therapeutic strategy targeted to minimize the risk of PAD. Autophagy is a catabolic process aimed at recycling damaged proteins and organelles. Cystathionine-γ-lyase (CSE) is a major H2S producing enzyme in the vasculature, which is vasoprotective. However, the role of CSE in regulating autophagy remains unknown. Hypothesis CSE derived sulfide metabolites protect endothelial cells from ischemic injury by inducing autophagy. Methods WT and CSE knockout mice were subjected to femoral artery ligation (FAL). Mouse aortic endothelial cells (MAECs) isolated from WT and CSE-/- mice served as in vitro model. Autophagic flux was measured with and without chloroquine (50mg/kg) and bafilomycin (50nM) in animals and cells respectively. Trypan blue assay was used to evaluate cell viability. Annexin V/ PI staining and the cleaved caspase-3 assay were used to evaluate apoptosis. Results In WT and CSE-/- mice subjected to FAL, CSE-/- mice showed defective autophagy. MAECs deficient in CSE-/- also showed impaired autophagy. Transfection with mRFP-GFP tandem fluorescent-tagged LC3 plasmid showed increased autolysosomes in WT compared to CSE-/- MAEC’s. Interestingly, re-expression of human CSE in CSE-/- endothelial cells restored autophagy. Exogenous sulfide treatment, including per/polysulfides were not able to restore autophagy in CSE-/- cells. However, lanthionine ketimine (LK), a novel sulfur metabolite of the transulfuration pathway, restored autophagy in its cell permeable ester form. Lastly, we observed that deficiency of CSE blocks autophagy leading to apoptosis in endothelial cells. Conclusion We reveal for the first time that LK, a CSE derived sulfur metabolite, could regulate autophagy in ischemic endothelial cells. These findings reveal a potential new experimental approach to protect against ischemic injury by activating autophagy during PAD.