Department of Surgery, University of Pittsburgh, Pittsburgh, PA, USA.
J Vasc Surg. 2012 Jan;55(1):180-91. doi: 10.1016/j.jvs.2011.07.072. Epub 2011 Sep 23.
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.
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).
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.
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.
骨骼肌缺血的血管生成驱动因素仍知之甚少。组织损伤和修复过程中会激活固有炎症途径，这表明这种高度保守的途径可能参与了缺血诱导的血管生成。我们假设，固有免疫信号的内源性配体之一，高迁移率族蛋白 B1（HMGB1），与缺氧或营养缺乏时的自噬反应相结合，在血管生成中发挥重要作用。
在常氧或低氧（1%氧气）条件下培养人真皮微血管内皮细胞（ECs）。进行 HMGB1 亚细胞定位的免疫细胞化学分析，评估管形成，并用 Western blot 分析肌球蛋白轻链 3I（LC3I）转化为 LC3II，作为自噬的标志物。用 3-甲基腺嘌呤（3MA）、氯喹或雷帕霉素分别抑制或促进自噬。在体内，进行小鼠后肢缺血模型。在 4 小时时采集肌肉样本以评估核 HMGB1，并在 14 天时检查内皮细胞密度。通过激光多普勒灌注成像（LDPI）计算后肢的灌注恢复。
与常氧细胞相比，低氧 ECs 的核 HMGB1 染色减少（平均荧光强度，186.9 ± 17.1 与 236.0 ± 1.6，P =.01），同时胞质染色增加。HMGB1 处理 ECs 增强了管形成，这是 ECs 的一种血管生成表型。内源性 HMGB1 的中和显着抑制管形成并抑制 LC3II 的形成。用 3MA 或氯喹抑制自噬会破坏管形成，而用雷帕霉素诱导自噬则增强管形成并促进 HMGB1 易位。在体内，与非缺血肌肉相比，缺血骨骼肌中的 HMGB1 阳性肌细胞核数量减少（34.9% ± 1.9%与 51.7% ± 2.0%，P <.001）。与对照后肢相比，将 HMGB1 注射到缺血后肢可使灌注恢复增加 21%，并使 EC 密度增加（49.2 ± 4.1 与 34.2 ± 3.4 个 EC/高倍视野，分别；P =.02）在 14 天时。
HMGB1 和自噬在 EC 中响应低氧或血清耗竭而发生核释放。HMGB1 和自噬是促进 EC 血管生成行为所必需的，并且可能相互依赖。在体内，HMGB1 可促进缺血损伤后的灌注恢复和 EC 密度增加。这些发现表明自噬和 HMGB1 在 EC 血管生成行为中可能存在潜在的机制联系，并支持固有免疫途径在血管生成中的重要性。
