Mirella Belleri

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.

The contribution of polymorphonuclear neutrophils (PMN) to host defense and natural immunity extends well beyond their traditional role as professional phagocytes. In this study, we demonstrate that upon stimulation with proinflammatory stimuli, human PMN release enzymatic activities that, in vitro, generate bioactive angiostatin fragments from purified plasminogen. We also provide evidence that these angiostatin-like fragments, comprising kringle domain 1 to kringle domain 3 (kringle 1-3) of plasminogen, are generated as a byproduct of the selective proteolytic activity of neutrophil-secreted elastase. Remarkably, affinity-purified angiostatin kringle 1-3 fragments generated by neutrophils inhibited basic fibroblast growth factor plus vascular endothelial growth factor-induced endothelial cell proliferation in vitro, and both vascular endothelial growth factor-induced angiogenesis in the matrigel plug assay and fibroblast growth factor-induced angiogenesis in the chick embryo chorioallantoic membrane assay, in vivo. These results represent the first demonstration that biologically active angiostatin-like fragments can be generated by inflammatory human neutrophils. Because angiostatin is a potent inhibitor of angiogenesis, tumor growth, and metastasis, the data suggest that activated PMN not only act as potent effectors of inflammation, but might also play a critical role in the inhibition of angiogenesis in inflammatory diseases and tumors, by generation of a potent anti-angiogenic molecule.

Angiogenesis plays a key role in various physiologic and pathologic conditions, including tumor growth. Drm/gremlin, a member the Dan family of bone morphogenic protein (BMP) antagonists, is commonly thought to affect different processes during growth, differentiation, and development by heterodimerizing various BMPs. Here, we identify Drm/gremlin as a novel proangiogenic factor expressed by endothelium. Indeed, Drm/gremlin was purified to homogeneity from the conditioned medium of transformed endothelial cells using an endothelial-cell sprouting assay to follow protein isolation. Accordingly, recombinant Drm/gremlin stimulates endothelial-cell migration and invasion in fibrin and collagen gels, binds with high affinity to various endothelial cell types, and triggers tyrosine phosphorylation of intracellular signaling proteins. Also, Drm/gremlin induces neovascularization in the chick embryo chorioallantoic membrane. BMP4 does not affect Drm/gremlin interaction with endothelium, and both molecules exert a proangiogenic activity in vitro and in vivo when administered alone or in combination. Finally, Drm/gremlin is produced by the stroma of human tumor xenografts in nude mice, and it is highly expressed in endothelial cells of human lung tumor vasculature when compared with non-neoplastic lung. Our observations point to a novel, previously unrecognized capacity of Drm/gremlin to interact directly with target endothelial cells and to modulate angiogenesis.