The chromosomal high mobility group box-1 (HMGB1) protein acts as a proinflammatory cytokine when released in the extracellular environment by necrotic and inflammatory cells. In the present study, we show that HMGB1 exerts proangiogenic effects by inducing MAPK ERK1/2 activation, cell proliferation, and chemotaxis in endothelial cells of different origin. Accordingly, HMGB1 stimulates membrane ruffling and repair of a mechanically wounded endothelial cell monolayer and causes endothelial cell sprouting in a three-dimensional fibrin gel. In keeping with its in vitro properties, HMGB1 stimulates neovascularization when applied in vivo on the top of the chicken embryo chorioallantoic membrane whose blood vessels express the HMGB1 receptor for advanced glycation end products (RAGE). Accordingly, RAGE blockade by neutralizing Abs inhibits HMGB1-induced neovascularization in vivo and endothelial cell proliferation and membrane ruffling in vitro. Taken together, the data identify HMGB1/RAGE interaction as a potent proangiogenic stimulus.

High mobility group proteins are chromatin binding factors with key roles in maintenance of nuclear homeostasis. The evidence indicates that extracellularly released high mobility group box 1 (HMGB1) protein behaves as a cytokine, promoting inflammation and participating to the pathogenesis of several disorders in peripheral organs. In this study, we have investigated the expression levels and relocation dynamics of HMGB1 in neural cells, as well as its neuropathological potential. We report that HMGB1 is released in the culture media of neurons and astrocytes challenged with necrotic but not apoptotic stimuli. Recombinant HMGB1 prompts induction of pro-inflammatory mediators such as inducible nitric oxide synthase (iNOS), cyclooxygenase-2, interleukin-1b, and tumor necrosis factor a, and increases excitotoxic as well as ischemic neuronal death in vitro. Dexamethasone reduces HMGB1 dependent immune glia activation, having no effect on the protein's neurotoxic effects. HMGB1 is expressed in the nucleus of neurons and astrocytes of the mouse brain, and promptly (1 h) translocates into the cytoplasm of neurons within the ischemic brain. Brain microinjection of HMGB1 increases the transcript levels of proinflammatory mediators and sensitizes the tissue to the ischemic injury. Together, data underscore the neuropathological role of nuclear HMGB1, and point to the protein as a mediator of post-ischemic brain damage.

Extracellular high-mobility group box 1 protein (HMGB1) triggers inflammatory events in the brain. We demonstrate that astrocytes, the main glial cells in the brain, acquire a specific reactive phenotype when exposed to HMGB1. This cell activation, which involves the receptor for advanced glycation end-products and the MAPK/ERK1/2 cascade, results in the transcriptional/translational induction of a restricted number of inflammatory mediators, including cyclooxygenase-2, matrix metalloproteinase-9, and several chemokines of the CC and CXC families. The mixture of factors released by HMGB1-reactive astrocytes displays a potent chemotactic activity on human monocytic cells. This study is the first to suggest that HMGB1/astrocyte interaction plays a specific functional role in the progression of inflammatory processes in the CNS by facilitating local leukocyte infiltration.

In the presence of micromolar concentrations of Ca2+, both protein kinase C and a cytosolic Ca2+-requiring neutral proteinase of human neutrophils become associated with the neutrophil membrane. Binding to the membrane results in activation of the proteinase, which then catalyzes limited proteolysis of the kinase to produce a form that is fully active in the absence of Ca2' and phospholipid. This irreversibly activated protein kinase is released from the membrane and may thus have access, in the intact cell, to intracellular protein substrates. In the absence of the proteinase, Ca2+ promotes the binding of protein kinase C, but conversion to the Ca2+/phospholipid-independent form does not occur and the kinase remains associated with the membrane fraction.Protein kinase C was originally described in rat brain as a soluble, cAMP-independent proenzyme (1) that was converted to the active kinase by the action of a cytosolic Ca2+-requiring proteinase (2, 3). The native "proenzyme" was later shown to require Ca2' and phospholipid (4,5) and to be further activated by diacylglycerol (6), which markedly increased its affinity for both Ca2' and phospholipid (for reviews, see refs. 7 and 8). Activation of protein kinase C in stimulated platelets has been attributed to the formation of diacylglycerol generated by phospholipase C from inositol phospholipids (7,9). An irreversible activation by limited proteolysis has also been described in platelets treated with phospholipase C (10) or phorbol 12-myristate 13-acetate (11) Isolation of Neutrophils. This was based on the procedure of Boyum (14). Freshly collected, heparinized human blood (100 ml) from healthy donors was treated with 1.6% (wt/vol) dextran (final concentration) and left at 25-28°C for -1 hr. The sedimented erythrocytes were removed and the supernatant solution (40 ml) was collected and layered onto 10 ml of 6% Ficoll 400 solution containing 0.17% (vol/vol) Urovison. The gradient was centrifuged at 800 x g for 20 min and the pellet obtained was resuspended in 10 ml of 0.2% NaCl to lyse the contaminating red cells. After 30 sec, 10 ml of 1.6% NaCl was added; the cells were recovered by centrifugation at 400 x g for 5 min and washed three times with 0.01 M sodium phosphate, pH 7.4/5 mM KCl/0.12 M NaCl/24 mM NaHCO3/5 mM glucose. Prior to use, the cells were maintained in an ice bath in the same medium at a concentration of 15-20 x 106 cells per ml. The cell population obtained consisted of 96% neutrophils, as evaluated by microscopic examination. The remaining 4% consisted of 3.5% eosinophils and 0.4% monocytes.Isolation of Human Platelets. Fresh human blood platelet concentrates were obtained from a blood bank and washed platelets were prepared as described by Baenziger and Majerus (15). The platelets were washed and suspended at a final concentration of 1010 cells per ml in the same buffer employed for the neutrophils.Isolation of the Soluble and Particulate Fractions from Neutrophils and Platelets. These were prepared from lysates obtained by sonicating ...

The multifunctional protein high mobility group box 1 (HMGB1) is expressed in hippocampus and cerebellum of adult mouse brain. Our aim was to determine whether HMGB1 affects glutamatergic transmission by monitoring neurotransmitter release from glial (gliosomes) and neuronal (synaptosomes) re-sealed subcellular particles isolated from cerebellum and hippocampus. HMGB1 induced release of the glutamate analogue [ 3 H]D-aspartate form gliosomes in a concentrationdependent manner, whereas nerve terminals were insensitive to the protein. The HMGB1-evoked release of [ 3 H]D-aspartate was independent of modifications of cytosolic Ca 2+ , but it was blocked by DL-threo-b-benzyloxyaspartate (DL-TBOA), an inhibitor of glutamate transporters. HMGB1 also stimulated the release of endogenous glutamate in a Ca 2+ -independent and DL-TBOA-sensitive manner. These findings suggest the involvement of carrier-mediated release. Moreover, dihydrokainic acid, a selective inhibitor of glutamate transporter 1 (GLT1), does not block the effect of HMGB1, indicating a role for the glial glutamate-aspartate transporter (GLAST) subtype in this response. We also demonstrate that HMGB1/glial particles association is promoted by Ca 2+ . Furthermore, although HMGB1 can physically interact with GLAST and the receptor for advanced glycation end products (RAGE), only its binding with RAGE is promoted by Ca 2+ . These results suggest that the HMGB1 cytokine could act as a modulator of glutamate homeostasis in adult mammal brain.