Over 60% of lower extremity amputations are performed in patients with diabetes and peripheral arterial disease, and at least 25% require subsequent reamputation due to poor surgical site healing. The mechanisms underlying poor amputation stump healing in the setting of diabetes are not understood. -acetylcysteine (NAC) is known to promote endothelial cell function and angiogenesis and may have therapeutic benefits in the setting of diabetes. We tested the hypothesis that NAC alters the vascular milieu to improve healing of amputation stumps in diabetes using a novel murine hindlimb ischemia-amputation model. Amputation stump tissue perfusion and healing were evaluated in C57BL/6J adult mice with streptozotocin-induced diabetes. Compared with controls, mice treated with daily NAC demonstrated improved postamputation stump healing, perfusion, adductor muscle neovascularization, and decreased muscle fiber damage. Additionally, NAC stimulated HUVEC migration and proliferation in a phospholipase C β-dependent fashion and decreased Gαq palmitoylation. Similarly, NAC treatment also decreased Gαq palmitoylation in ischemic and nonischemic hindlimbs In summary, we demonstrate that NAC accelerates healing of amputation stumps in the setting of diabetes and ischemia. The underlying mechanism appears to involve a previously unrecognized effect of NAC on Gαq palmitoylation and phospholipase C β-mediated signaling in endothelial cells.-Zayed, M. A., Wei, X., Park, K., Belaygorod, L., Naim, U., Harvey, J., Yin, L., Blumer, K., Semenkovich, C. F.-acetylcysteine accelerates amputation stump healing in the setting of diabetes.
The endothelial cell (EC) lipid microenvironment can directly impact post-translational lipid modification of critical intracellular signaling proteins, which has important vascular disease implications. The antioxidant N-Acetyl-Cysteine (NAC) is a well-known thiol that regulates intracellular glutathione levels, activates various intracellular kinases, and promotes EC proliferation. In erythrocytes, NAC alters cellular lipid content and modulates generation of plasma membrane phosphatidylserine (PS). To test the hypothesis that NAC similarly alters the lipid microenvironment in ECs, we evaluated the effect of NAC on S(cysteine)-protein lipidation, as well as its effects on cellular lipid content in HUVECs. Increasing the duration of NAC treatment (from 2 to 16 hours) and increasing the concentration of NAC (from 1 to 5 mM) robustly decreased S-protein lipidation of the heterotrimeric G protein subunit, Gaq, by 80% (p<0.05) without affecting Gaq protein levels. There was no effect of NAC on lipidation of the Caveolin-1, a lipid raft protein known to be involved in vascular function. NAC also suppressed expression (by 55%, p<0.05) of Fatty Acid Synthase (FAS), a critical lipogenic enzyme implicated in generating palmitate utilized for the lipidation of vascular proteins. Since Gaq-mediated regulation of downstream phospholipases affects cytosolic phospholipids, we characterized lipidomic profiles of NAC-treated ECs using electrospray ionization mass spectrometry analysis. Preliminary findings demonstrated that treatment with NAC 5mM alters global PS and phosphatidylcholine (PC) content in HUVECs, with a 160% increase in total PS lipid species and a 140% increase in total PC lipid species. These findings suggest that NAC remodels the lipid milieu of endothelial cells in a manner likely to impact signaling pathways relevant to inflammation, proliferation, and repair in the vasculature.
Over 150,000 extremity amputations are performed in the US per year. More than 60% of these are performed in patients with diabetes and peripheral arterial disease, and 25% require subsequent re-amputation due to poor surgical site healing. The mechanisms underlying poor amputation stump healing in the setting of diabetes are not understood, which greatly limits management options. N-acetylcysteine (NAC) is a well-known thiol that regulates intracellular glutathione levels, promotes endothelial cell function and angiogenesis, and is proposed to have therapeutic benefits in the setting of diabetes. To test the hypothesis that NAC can improve surgical site healing in chronically ischemic amputation stumps in the setting of diabetes, we developed a novel in vivo murine hindlimb ischemia-amputation model. In this model, streptozotocin-treated C57B6 mice are allowed to develop hyperglycemia for 1 month, followed by unilateral hindlimb femoral artery ligation. After 1 week, the recovered yet still mal-perfused hindlimb undergoes an infrageniculate amputation. Compared to sham control, mice receiving a daily intraperitoneal administration of NAC 150mg/kg demonstrated a 31% improvement in stump healing (P=0.06), 160% increase in hindlimb stump laser-Doppler perfusion at 3 days post-amputation (P=0.03), as well as 179% increase in hindlimb adductor muscle neovascularization (P=0.01). Adductor muscle segments from ischemic amputated hindlimbs also demonstrated less muscle damage, and expressed higher levels of de-palmitoylated activated Gaq (P<0.05), which is essential for ischemia-induced neovascularization. Similarly, HUVECs treated with NAC 3mM demonstrated a transient 19% increase in de-palmitoylated Gaq (P=0.03), suggesting a novel mechanism of action for NAC. These findings demonstrate that NAC may have important therapeutic benefits in amputation stump healing in the setting of diabetes, which may translate into a valuable treatment option in vulnerable patient populations.
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