An angiogenic role for the human peptide antibiotic LL-37/hCAP-18.

Antimicrobial peptides are effector molecules of the innate immune system and contribute to host defense and regulation of inflammation. The human cathelicidin antimicrobial peptide LL-37/hCAP-18 is expressed in leukocytes and epithelial cells and secreted into wound and airway surface fluid. Here we show that LL-37 induces angiogenesis mediated by formyl peptide receptor-like 1 expressed on endothelial cells. Application of LL-37 resulted in neovascularization in the chorioallantoic membrane assay and in a rabbit model of hind-limb ischemia. The peptide directly activates endothelial cells, resulting in increased proliferation and formation of vessel-like structures in cultivated endothelial cells. Decreased vascularization during wound repair in mice deficient for CRAMP, the murine homologue of LL-37/hCAP-18, shows that cathelicidin-mediated angiogenesis is important for cutaneous wound neovascularization in vivo. Taken together, these findings demonstrate that LL-37/hCAP-18 is a multifunctional antimicrobial peptide with a central role in innate immunity by linking host defense and inflammation with angiogenesis and arteriogenesis.

VEGFR-1-selective VEGF homologue PlGF is arteriogenic: evidence for a monocyte-mediated mechanism.

Two signaling receptors for vascular endothelial growth factor (VEGF) in the vasculature are known with not yet well-understood roles in collateral vessel growth (arteriogenesis). In this study, we examined the involvement of the two VEGF receptors in arteriogenesis. Therefore, we used the VEGF homologue placenta growth factor (PlGF), which only binds to VEGFR-1 and VEGF-E, which only recognizes VEGFR-2. These peptides were locally infused over 7 days after ligation of the femoral artery in the rabbit. Evaluation of collateral growth by determining collateral conductance and angiographic scores demonstrated that the VEGFR-1-specific PlGF contributed significantly more to arteriogenesis than the VEGFR-2 specific VEGF-E. The combination of VEGF-E and PlGF did not exceed the effect of PlGF alone, indicating that cooperation of the two VEGF receptors in endothelial cell signaling is not required for arteriogenesis. In an in vitro model of angiogenesis, VEGF and VEGF-E were comparably active, whereas PlGF displayed no activity when given alone and did not further increase the effects of VEGF or VEGF-E. However, PlGF was as potent as VEGF when monocyte activation was assessed by monitoring integrin surface expression. In addition, accumulation of activated monocytes/macrophages in the periphery of collateral vessels in PlGF-treated animals was observed. Furthermore, in monocyte-depleted animals, the ability of PlGF to enhance collateral growth in the rabbit model and to rescue impaired arteriogenesis in PlGF gene-deficient mice was abrogated. Together, these data indicate that the arteriogenic activity observed with the VEGFR-1-specific PlGF is caused by its monocyte-activating properties.

NAB2, a corepressor of EGR-1, inhibits vascular endothelial growth factor-mediated gene induction and angiogenic responses of endothelial cells.

In this study we have investigated the role of a specific corepressor of EGR-1, NAB2, to down-regulate vascular endothelial growth factor (VEGF)-induced gene expression in endothelial cells and to inhibit angiogenesis. Firstly, we show a reciprocal regulation of EGR-1 and NAB2 following VEGF treatment. During the initial phase EGR-1 is rapidly induced and NAB2 levels are down-regulated. This is followed by a reduction of EGR-1 and a concomitant increase of NAB2. Secondly, using the tissue factor gene as a readout for VEGF-induced and EGR-1-regulated gene expression we demonstrate that NAB2 can completely block VEGF-induced tissue factor reporter gene activity. Thirdly, by adenovirus-mediated expression we show that NAB2 inhibits up-regulation of tissue factor, VEGF receptor-1, and urokinase plasminogen activator mRNAs even when a combination of VEGF and bFGF is used for induction. In addition, NAB2 overexpression significantly reduced tubule and sprout formation in two different in vitro angiogenesis assays and largely prevented the invasion of cells and formation of vessel-like structures in the murine Matrigel model. These data suggest that NAB2 regulation represents a mechanism to guarantee transient EGR-1 activity following exposure of endothelial cells to VEGF and that NAB2 overexpression could be used to inhibit signals involved in the early phase of angiogenesis.

p38 MAP kinase--a molecular switch between VEGF-induced angiogenesis and vascular hyperpermeability.

Vascular endothelial growth factor (VEGF) is not only essential for vasculogenesis and angiogenesis but also is a potent inducer of vascular permeability. Although a dissection of the molecular pathways between angiogenesis- and vascular permeability-inducing properties would be desirable for the development of angiogenic and anti-angiogenic therapies, such mechanisms have not been identified yet. Here we provide evidence for a role of the p38 MAPK as the signaling molecule that separates these two processes. Inhibition of p38 MAPK activity enhances VEGF-induced angiogenesis in vitro and in vivo, a finding that was accompanied by prolonged Erk1/2 MAPK activation, increased endothelial survival, and plasminogen activation. Conversely, the same inhibitors abrogate VEGF-induced vascular permeability in vitro and in vivo. These dualistic properties of p38 MAPK are relevant not only for therapeutic angiogenesis but also for reducing edema formation and enhancing tissue repair in ischemic diseases.

An engineered heparin-binding form of VEGF-E (hbVEGF-E). Biological effects in vitro and mobilizatiion of precursor cells.

Vascular endothelial growth factor (VEGF-A) is the founding member of a family of angiogenic proteins with various binding abilities to three cognate VEGF receptors. Previously, a gene encoding from the genome of parapox orf virus (OV) with about 25% amino acid identity to mammalian VEGF-A was named VEGF-E and shown to bind and specifically activate the vascular endothelial growth factor receptor VEGFR-2 (KDR/flk-1). Here, we have generated a novel heparin-binding form of VEGF-E by introducing the heparin-domain of the human VEGF-A(165) splice variant into the viral VEGF-E protein. Recombinant heparin-binding VEGF-E (hbVEGF-E) is shown to stimulate proliferation and sprout formation of macro- and microvascular endothelial cells to a similar extent as the parental OV-VEGF-E but fails to activate peripheral mononuclear cells. However, hbVEGF-E is more potent in binding competition assays with primary human endothelial cells when compared to the OV-VEGF-E. This can be explained by our finding that binding of hbVEGF-E but not of parental OV-VEGF-E to the VEGFR-2 is strongly increased by the addition of neuropilin-1 (NP-1), a cognate co-receptor for VEGF-A. The engineered hbVEGF-E was compared with the VEGFR-1 selective and also heparin-binding form of placenta growth factor (PlGF-2) in vivo. Both heparin-binding homologues induced mobilization of endothelial progenitor cells from the bone marrow and gave rise to similar colony numbers of myeloic cells in a colony-forming assay. These findings suggest that both VEGFR-1 and VEGFR-2 are involved in stem cell mobilization.