title: Chloroquine improves the response to ischemic muscle injury and increases HMGB1 after arterial ligation.
abstract: 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.
  
  
  
  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.
  
  
  
  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.
  
  
  
  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.
authors.affiliation: Department of Surgery, University of Pittsburgh Medical Center, Pittsburgh, Pa.
authors.forename: Jun
authors.initials: J
authors.lastname: Xu
authors.affiliation: Department of Surgery, University of Pittsburgh Medical Center, Pittsburgh, Pa.
authors.forename: Xiangdong
authors.initials: X
authors.lastname: Cui
authors.affiliation: Department of Surgery, University of Pittsburgh Medical Center, Pittsburgh, Pa.
authors.forename: Jiehua
authors.initials: J
authors.lastname: Li
authors.affiliation: Department of Surgery, University of Nebraska Medical Center, Omaha, Neb.
authors.forename: Panagiotis
authors.initials: P
authors.lastname: Koutakis
authors.affiliation: Department of Surgery, University of Nebraska Medical Center, Omaha, Neb.
authors.forename: Iraklis
authors.initials: I
authors.lastname: Pipinos
authors.affiliation: Department of Surgery, University of Pittsburgh Medical Center, Pittsburgh, Pa; Department of Veterans Affairs, Pittsburgh, Pa.
authors.forename: Edith
authors.initials: E
authors.lastname: Tzeng
authors.affiliation: Department of Surgery, University of Pittsburgh Medical Center, Pittsburgh, Pa.
authors.forename: Alex
authors.initials: A
authors.lastname: Chen
authors.affiliation: Department of Surgery, University of Pittsburgh Medical Center, Pittsburgh, Pa. Electronic address: sachdevu2@upmc.edu.
authors.forename: Ulka
authors.initials: U
authors.lastname: Sachdev
mesh_terms: D000368:Aged; D000818:Animals; D001343:Autophagy / Q000187:drug effects; D001783:Blood Flow Velocity; D016022:Case-Control Studies; D020170:Caspase 1 / Q000378:metabolism; D002460:Cell Line; D002738:Chloroquine / Q000494:pharmacology*; D004195:Disease Models, Animal; D005260:Female; D005263:Femoral Artery / Q000601:surgery*; D024243:HMGB1 Protein / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007383:Intermittent Claudication / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D007511:Ischemia / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008026:Ligation; D008297:Male; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008875:Middle Aged; D018482:Muscle, Skeletal / Q000098:blood supply* / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D058729:Peripheral Arterial Disease / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D020127:Recovery of Function; D012039:Regional Blood Flow; D000071456:Sequestosome-1 Protein / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D015854:Up-Regulation
chemical_list: D024243:HMGB1 Protein; C585491:HMGB1 protein, human; C582808:HMGB1 protein, mouse; D000071456:Sequestosome-1 Protein; C101343:Sqstm1 protein, mouse; D002738:Chloroquine; D007770:L-Lactate Dehydrogenase; D020170:Caspase 1