OBJECTIVE: We have previously shown that exogenous administration of the nuclear 
protein high mobility group box 1 (HMGB1) improves angiogenesis after tissue 
ischemia. Antagonizing HMGB1 prolongs muscle necrosis and deters regeneration. 
In this study, we evaluated HMGB1 expression in peripheral arterial disease 
(PAD) and the mechanisms that promote its release in a murine model of hindlimb 
ischemia. Specifically, we investigated how chloroquine (CQ), a commonly 
employed disease-modifying antirheumatic drug, promotes HMGB1 release from 
muscle. We hypothesized that CQ could increase HMGB1 locally and systemically, 
allowing it to mediate recovery from ischemic injury.
METHODS: Muscle biopsies were performed on patients undergoing lower extremity 
surgery for non-PAD-related disease as well as for claudication and critical 
limb ischemia. Clinical symptoms and ankle-brachial indices were recorded for 
each patient. HMGB1 was detected in muscle sections using immunohistochemical 
staining. Unilateral femoral artery ligation was performed on both wild-type and 
inducible HMGB1 knockout mice. Wild-type mice were administered intraperitoneal 
CQ 2 weeks before and after femoral artery ligation. Laser Doppler perfusion 
imaging was used to determine perfusion recovery. Serum and tissue levels of 
HMGB1 were measured at designated time points. In vitro, cultured C2C12 
myoblasts were treated with increasing doses of CQ. HMGB1, autophagosome 
formation, p62/SQSTM1 accumulation, caspase-1 expression and activity, and 
lactate dehydrogenase levels were measured in supernatants and cell lysates.
RESULTS: Nuclear expression of HMGB1 was prominent in patients with claudication 
and critical limb ischemia (P < .05) compared with controls. CQ-treated mice had 
elevated serum HMGB1 and diffuse HMGB1 staining in muscle (P < .01). In 
wild-type mice, CQ treatment resulted in higher laser Doppler perfusion imaging 
ratios in the ischemic limb at 7 days (P < .03) and less fat replacement after 
2 weeks (P < .03). In cultured myoblasts, CQ induced autophagosome accumulation, 
inhibited p62/SQSTM-1 degradation, and activated caspase-1.
CONCLUSIONS: HMGB1 is prominently expressed in PAD muscle but mostly confined to 
the nucleus. Our in vivo data suggest that HMGB1 mobilization into the 
sarcoplasm and serum can be increased with CQ, possibly through 
caspase-1-mediated pathways. Whereas HMGB1 can be released by many cell types, 
these studies suggest that the muscle may be an important additional source that 
is relevant in PAD.