Introduction: Peripheral arterial disease (PAD) is a chronic arterial occlusive disease leading to lower limb ischemia. Autophagy is a cell adaptive response associated with ischemic stress that may act as a double edged sword. It is essential during tissue ischemia, but may also phagocytize more organelles promoting cell death. Hydrogen sulfide (H2S), a gasotransmitter predominantly produced in the vasculature via cystathionine-γ-lyase (CSE), play a key regulatory role in the cardiovascular functions. However, role of H2S/CSE in autophagy, and its associated impact on ischemic injury is poorly understood.
Objective: To investigate the role of CSE/H2S in the regulation of autophagy under ischemia using murine hind limb ischemia model and in vitro studies.
Methods: Unilateral femoral artery ligation (FAL) was performed on C57BL/6J(WT) mice and tissue blood perfusion was measured using laser Doppler flowmetry. LC3B expression was measured in the skeletal muscle tissues by immunohistochemistry. We utilized mouse aortic endothelial cells (MAECs) isolated from WT and CSE deficient (CSEKO) mice respectively and immortalized them. We treated WT and CSE KO MAECs were subjected to normoxia and hypoxia with and without autophagy inhibitor bafilomycin and evaluated for LC3B. ROS levels were evaluated by DCFDH. Apoptosis was measured by LDH assay and cleaved caspase-3.
Results: Compared to WT, CSEKO MAECs showed significant reduction in LC3B protein expression. Reduced LC3B and p62 mRNA expressions were also observed in CSEKO cells. In the WT mice subjected to FAL, blood perfusion was significantly reduced in the ligated limbs compared to non-ligated. Interestingly, LC3B was significantly higher in ischemic muscle compared to non-ischemic tissues. Additionally, CSE genetic deletion significantly increased ROS levels that lead to increased apoptosis. CSEKO MAECs showed increased LDH activity and elevated cleaved caspase-3 expression under hypoxia.
Conclusions: Our study reveals for the first time that CSE/H2S regulates autophagy in ischemic endothelial cells. CSE/H2S may be used as a therapeutic agent to protect against ischemia injury by inhibiting autophagy during PAD.