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.
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