title: High mobility group box 1 promotes endothelial cell angiogenic behavior in vitro and improves muscle perfusion in vivo in response to ischemic injury.
abstract: OBJECTIVE
  The angiogenic drive in skeletal muscle ischemia remains poorly understood. Innate inflammatory pathways are activated during tissue injury and repair, suggesting that this highly conserved pathway may be involved in ischemia-induced angiogenesis. We hypothesize that one of the endogenous ligands for innate immune signaling, high mobility group box 1 (HMGB1), in combination with autophagic responses to hypoxia or nutrient deprivation, plays an important role in angiogenesis.
  
  
  METHODS
  Human dermal microvascular endothelial cells (ECs) were cultured in normoxia or hypoxia (1% oxygen). Immunocytochemical analysis of HMGB1 subcellular localization, evaluation of tube formation, and Western blot analysis of myotubule light-chain 3I (LC3I) conversion to LC3II, as a marker of autophagy, were conducted. 3-Methyladenine (3MA), chloroquine, or rapamycin were administered to inhibit or promote autophagy, respectively. In vivo, a murine hind limb ischemia model was performed. Muscle samples were collected at 4 hours to evaluate for nuclear HMGB1 and at 14 days to examine endothelial density. Perfusion recovery in the hind limbs was calculated by laser Doppler perfusion imaging (LDPI).
  
  
  RESULTS
  Hypoxic ECs exhibited reduced nuclear HMGB1 staining compared with normoxic cells (mean fluorescence intensity, 186.9 ± 17.1 vs 236.0 ± 1.6, P = .01) with a concomitant increase in cytosolic staining. HMGB1 treatment of ECs enhanced tube formation, an angiogenic phenotype of ECs. Neutralization of endogenous HMGB1 markedly impaired tube formation and inhibited LC3II formation. Inhibition of autophagy with 3MA or chloroquine abrogated tube formation, whereas its induction with rapamycin enhanced tubing and promoted HMGB1 translocation. In vivo, ischemic skeletal muscle showed reduced numbers of HMGB1-positive myocyte nuclei compared with nonischemic muscle (34.9% ± 1.9% vs 51.7% ± 2.0%, P < .001). Injection of HMGB1 into ischemic hind limbs increased perfusion recovery by 21% and increased EC density (49.2 ± 4.1 vs 34.2 ± 3.4 ECs/high-powered field, respectively; P = .02) at 14 days compared with control hind limbs.
  
  
  CONCLUSIONS
  Nuclear release of HMGB1 and autophagy occur in ECs in response to hypoxia or serum depletion. HMGB1 and autophagy are necessary and likely play an interdependent role in promoting the angiogenic behavior of ECs. In vivo, HMGB1 promotes perfusion recovery and increased EC density after ischemic injury. These findings suggest a possible mechanistic link between autophagy and HMGB1 in EC angiogenic behavior and support the importance of innate immune pathways in angiogenesis.
authors.affiliation: Department of Surgery, University of Pittsburgh, Pittsburgh, PA, USA.
authors.forename: Ulka
authors.initials: U
authors.lastname: Sachdev
authors.forename: Xiangdong
authors.initials: X
authors.lastname: Cui
authors.forename: Guiying
authors.initials: G
authors.lastname: Hong
authors.forename: Seung
authors.initials: S
authors.lastname: Namkoong
authors.forename: Jenny M
authors.initials: JM
authors.lastname: Karlsson
authors.forename: Catherine J
authors.initials: CJ
authors.lastname: Baty
authors.forename: Edith
authors.initials: E
authors.lastname: Tzeng
mesh_terms: D043925:Angiogenesis Inducing Agents / Q000008:administration & dosage; D020533:Angiogenesis Inhibitors / Q000494:pharmacology; D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D001343:Autophagy; D015153:Blotting, Western; D015687:Cell Hypoxia; D004195:Disease Models, Animal; D042783:Endothelial Cells / Q000187:drug effects / Q000276:immunology / Q000378:metabolism* / Q000473:pathology; D024243:HMGB1 Protein / Q000008:administration & dosage / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006614:Hindlimb; D006801:Humans; D007113:Immunity, Innate; D007273:Injections, Intramuscular; D007511:Ischemia / Q000188:drug therapy / Q000276:immunology / Q000378:metabolism* / Q000473:pathology / Q000503:physiopathology; D017078:Laser-Doppler Flowmetry; D008297:Male; D051379:Mice; D008809:Mice, Inbred C3H; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D018485:Muscle Fibers, Skeletal / Q000187:drug effects / Q000276:immunology / Q000378:metabolism* / Q000473:pathology; D018482:Muscle, Skeletal / Q000098:blood supply* / Q000187:drug effects / Q000276:immunology / Q000378:metabolism* / Q000473:pathology; D018919:Neovascularization, Physiologic / Q000187:drug effects; D010100:Oxygen / Q000378:metabolism; D021381:Protein Transport; D012039:Regional Blood Flow; D013997:Time Factors
chemical_list: D043925:Angiogenesis Inducing Agents; D020533:Angiogenesis Inhibitors; D000906:Antibodies; D024243:HMGB1 Protein; C477012:MAP1LC3A protein, human; D008869:Microtubule-Associated Proteins; D010100:Oxygen