Abstract 16856: S-Glutathionylation of Endothelial Cell Proteins Facilitates Ischemic Limb Revascularization

Abstract

Reactive oxygen species (ROS) are increased during ischemia, but decreasing ROS in endothelial cells impairs revascularization. Low levels of ROS regulate protein function by inducing S-glutathionylation, a reversible post-translational protein modification that may change protein(s) function directly. Protein S-glutathionylation is increased in oxidative conditions and eliminated by glutaredoxin-1 (Glrx). We demonstrated that hind limb revascularization was impaired in global Glrx transgenic mice. In this study, we further examined the role of S-glutathionylation in revascularization by gain and loss of cell-specific Glrx expression. Methods: Hind limb ischemia (HLI) was created by femoral artery ligation. HLI surgery was performed in 1) “Tet-Off” endothelial specific Glrx transgenic mice (EC-Glrx TG), 2) global Glrx knockout (KO) mice, 3) myeloid cell-specific Glrx deleted mice. EC-Glrx TG was generated by breeding VEcadherin-tTA and tet-operated Glrx mice, treated with doxycycline in utero and until 8 weeks of age to avoid overexpression during development. 3) Myeloid cell-specific Glrx deleted mice were generated by bone marrow transplant using Glrx KO and wild type bone marrow, respectively. Blood flow recovery was analyzed by LASER Döppler. Result: S-glutathionylation of ischemic limb was increased more than two-fold compared with non-ischemic limb. S-glutathionylation was decreased in endothelial cells from EC-Glrx TG and increased in Glrx KO cells. 1) Blood flow recovery was significantly impaired in EC-Glrx TG mice compared with wild type littermate 1 week after HLI surgery (12% vs 39%, p<0.01), and 40% of ischemia limbs became necrotic and were lost in 2 weeks in EC-Glrx TG mice, while all limbs survived in WT mice. In contrast, 2) Blood flow recovery was improved in Glrx KO mice (57% in KO mice vs 42% in WT mice, p<0.01), 3). Myeloid-specific deletion of Glrx did not alter the blood flow recovery. Conclusions: Glrx in endothelial cells, not in myeloid cells, plays an important role in ischemic revascularization. Our data suggest that ROS-induced S-glutathionylation in EC positively regulates revascularization after limb ischemia.