High mobility group box 1 promotes endothelial cell angiogenic behavior in vitro and improves muscle perfusion in vivo in response to ischemic injury

Abstract

Objectives 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 (EC) 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 3 (LC3I) conversion to LC3II, as a marker of autophagy, were conducted. 3-methyladenine (3MA), chloroquine (CQ), 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 EC exhibited reduced nuclear HMGB1 staining compared with normoxic cells (mean fluorescence intensity 186.9 ± 17.1 vs. 236.0 ± 1.6, respectively, P = 0.01) 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 CQ abrogated tube formation while its induction with rapamycin enhanced tubing and promoted HMGB1 translocation. In vivo, ischemic skeletal muscle showed reduced the numbers of HMGB1 positive myocyte nuclei compared with nonischemic muscle (34.9% ± 1.9 vs. 51.7% ± 2.0, respectively, P<0.001). Injection of HMGB1 into ischemic hind-limbs increased perfusion recovery by 21% and increased EC density (49.2 ± 4.1vs. 34.2 ± 3.4 EC/HPF, respectively; p=0.02) at 14 days compared to control treated hind-limbs. Conclusion 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 are the first to suggest a possible mechanistic link between autophagy and HMGB1 in EC angiogenic behavior and support the importance of innate immune pathways in angiogenesis.

High Mobility Group Box 1 Inhibits Human Pulmonary Artery Endothelial Cell Migration via a Toll-like Receptor 4- and Interferon Response Factor 3-dependent Mechanism(s)

“…Hypoxia Induces HMGB1 Release from HPAECs-Several groups have previously shown that exposure to acute hypoxia leads to the release of HMGB1 from a variety of cell types, including systemic endothelial cells (6,21). It has, however, not been explored in pulmonary endothelial cells.…”

“…In particular, HMGB1 has been shown to stimulate cell migration and angiogenesis in HUVECs (6,(22)(23)(24). We were, therefore, curious to know whether we could recapitulate these results.…”

“…The importance of HMGB1 as a proinflammatory cytokine has been shown in diseases of chronic inflammation and exaggerated immune responses, such as atherosclerosis, and sepsis (4). HMGB1 has also been identified as a proangiogenic cytokine involved in tumor-and ischemia-induced angiogenesis (5)(6)(7)(8).…”

Messengers without Borders

“…[69][70][71][72] In addition, HMGB1 is reported to possess proangiogenic activity. [73][74][75][76] Its ability to promote proliferation, migration, and differentiation of several cell types, particularly those involved in angiogenesis, prompted us to examine the ability of HMGB1 to mobilize EPCs from the bone marrow. We observed significant mobilization of bone marrow EPCs and their initial sequestration in the spleen.…”

TLR4 Deters Perfusion Recovery and Upregulates Toll-like Receptor 2 (TLR2) in Ischemic Skeletal Muscle and Endothelial Cells

“…Whereas most have focused on the identification and testing of proangiogenic compounds or cellular therapies, there has been much less emphasis on evaluating the role of modulating inflammation to improve tissue recovery from the ischemic insult. We and others have investigated the role of toll-like receptor (TLR) activation in promoting angiogenesis and muscle recovery in a murine model of hindlimb ischemia (7)(8)(9)(10)(11)(12)(13)(14). TLRs mediate innate immune responses to bacterial components, including gram-negative bacterial lipopolysaccharide (LPS) (TLR4) and gram-positive bacterial lipoproteins (TLR2) (15)(16)(17)(18).…”