Matthias Clauss

The Vascular Endothelial Growth Factor Receptor Flt-1 Mediates Biological Activities IMPLICATIONS FOR A FUNCTIONAL ROLE OF PLACENTA GROWTH FACTOR IN MONOCYTE ACTIVATION AND CHEMOTAXIS

Two distinct receptors for vascular endothelial growth factor (VEGF), the tyrosine kinase receptors Flt-1 and Flk-1/KDR, have been described. In this study we show that monocytes, in contrast to endothelium, express only the VEGF receptor Flt-1, and that this receptor specifically binds also the VEGF homolog placenta growth factor (PlGF). Both VEGF and PlGF stimulate tissue factor production and chemotaxis in monocytes at equivalent doses. In contrast, endothelial cells expressing both the Flt-1 and the Flk-1/KDR receptors produce more tissue factor upon stimulation with VEGF than after stimulation with PlGF. Neutralizing antibodies to the KDR receptor reduce the VEGF-stimulated tissue factor induction in endothelial cells to levels obtained by stimulation with PlGF alone, but do not affect PlGF-induced tissue factor induction in endothelial cells nor the VEGF-dependent tissue factor production in monocytes. These findings strongly suggest Flt-1 as a functional receptor for VEGF and PlGF in monocytes and endothelial cells and identify this receptor as a mediator of monocyte recruitment and procoagulant activity.

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.

Inhibition of apoptosis induced by ischemia-reperfusion prevents inflammation.

Ischemia followed by reperfusion leads to severe organ injury and dysfunction. Inflammation is considered to be the most important cause of tissue injury in organs subjected to ischemia. The mechanism that triggers inflammation and organ injury after ischemia remains to be elucidated, although different causes have been postulated. We investigated the role of apoptosis in the induction of inflammation and organ damage after renal ischemia. Using a murine model, we demonstrate a relationship between apoptosis and subsequent inflammation. At the time of reperfusion, administration of the antiapoptotic agents IGF-1 and ZVAD-fmk (a caspase inactivator) prevented the early onset of not only renal apoptosis, but also inflammation and tissue injury. Conversely, when the antiapoptotic agents were administered after onset of apoptosis, these protective effects were completely abrogated. The presence of apoptosis was directly correlated with posttranslational processing of the endothelial monocyte-activating polypeptide II (EMAP-II), which may explain apoptosis-induced influx and sequestration of leukocytes in the reperfused kidney. These results strongly suggest that apoptosis is a crucial event that can initiate reperfusion-induced inflammation and subsequent tissue injury. The newly described pathophysiological insights provide important opportunities to effectively prevent clinical manifestations of reperfusion injury in the kidney, and potentially in other organs.

A novel vascular endothelial growth factor encoded by Orf virus, VEGF-E, mediates angiogenesis via signalling through VEGFR-2 (KDR) but not VEGFR-1 (Flt-1) receptor tyrosine kinases

The different members of the vascular endothelial growth factor (VEGF) family act as key regulators of endothelial cell function controlling vasculogenesis, angiogenesis, vascular permeability and endothelial cell survival. In this study, we have functionally characterized a novel member of the VEGF family, designated VEGF‐E. VEGF‐E sequences are encoded by the parapoxvirus Orf virus (OV). They carry the characteristic cysteine knot motif present in all mammalian VEGFs, while forming a microheterogenic group distinct from previously described members of this family. VEGF‐E was expressed as the native protein in mammalian cells or as a recombinant protein in Escherichia coli and was shown to act as a heat‐stable, secreted dimer. VEGF‐E and VEGF‐A were found to possess similar bioactivities, i.e. both factors stimulate the release of tissue factor (TF), the proliferation, chemotaxis and sprouting of cultured vascular endothelial cells in vitro and angiogenesis in vivo. Like VEGF‐A, VEGF‐E was found to bind with high affinity to VEGF receptor‐2 (KDR) resulting in receptor autophosphorylation and a biphasic rise in free intracellular Ca2+ concentration, whilst in contrast to VEGF‐A, VEGF‐E did not bind to VEGF receptor‐1 (Flt‐1). VEGF‐E is thus a potent angiogenic factor selectively binding to VEGF receptor‐2. These data strongly indicate that activation of VEGF receptor‐2 alone can efficiently stimulate angiogenesis.

VEGFR-1–Selective VEGF Homologue PlGF Is Arteriogenic: Evidence for a Monocyte-Mediated Mechanism

Abstract— 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.

Specificity, diversity, and convergence in VEGF and TNF-α signaling events leading to tissue factor up-regulation via EGR-1 in endothelial cells

Tissue factor (TF) has been shown to be up‐regulated in endothelial cells by the inflammatory cytokine tumor necrosis factor a (TNF‐α) as well as by the main angiogenic factor VEGF. Since both stimuli induce the transcription factor EGR‐1, which is critically involved in TF gene regulation, we used EGR‐1‐dependent TF induction as a model to identify potential cross‐talks between the various signal transduction cascades initiated by VEGF and TNF‐α. The data show that at the MAP kinase level, VEGF mainly activates ERK1/2 and p38 MAP kinases in human endothelial cells. TNF‐α is able to activate all three MAP kinase cascades as well as the classical inflammatory IκB/NFκB pathway. Furthermore, the MEK/ERK module of MAP kinases appears to act as the convergence point of VEGF‐ and TNF‐α‐initiated signaling cascades, which lead to the activation of EGR‐1 and subsequent TF expression, whereas the upstream signals are distinct. We found that induction of TF by VEGF via EGR‐1 is strongly PKC dependent. The TNF‐α‐initiated MEK/ERK cascade connected to EGR‐1 and TF expression is clearly less sensitive to PKC inhibition. TNF‐α‐mediated activation of MEK/ERK and EGR‐1 can be blocked by adenoviral expression of a dominant negative mutant of IKK2, whereas the VEGF signaling pathway is unaffected. Thus, our data demonstrate a new link between the classical inflammatory IKK/IκB and the MEK/ERK cascades triggered by TNF‐α. The additional finding that EGF induces ERK and EGR‐1 in a PKC independent manner and that this signal is not sufficient to up‐regulate TF emphasizes the importance of a VEGF‐specific signaling pattern for the induction of TF.— Mechtcheriakova, D., Schabbauer, G., Lucerna, M., Clauss, M., de Martin, R., Binder, B. R., Hofer, E. Specificity, diversity, and convergence in VEGF and TNF‐α signaling events leading to tissue factor up‐regulation via EGR‐1 in endothelial cells. FASEB J. 15, 230–242 (2001)

Hypoxia induces permeability in brain microvessel endothelial cells via VEGF and NO

In this study, an in vitro model of the blood-brain barrier, consisting of porcine brain-derived microvascular endothelial cells (BMEC), was used to evaluate the mechanism of hypoxia-induced hyperpermeability. We show that hypoxia-induced permeability in BMEC was completely abolished by a neutralizing antibody to vascular endothelial growth factor (VEGF). In contrast, under normoxic conditions, addition of VEGF up to 100 ng/ml did not alter monolayer barrier function. Treatment with either hypoxia or VEGF under normoxic conditions induced a twofold increase in VEGF binding sites and VEGF receptor 1 (Flt-1) mRNA expression in BMEC. Hypoxia-induced permeability also was prevented by the nitric oxide (NO) synthase inhibitor NG-monomethyl-L-arginine, suggesting that NO is involved in hypoxia-induced permeability changes, which was confirmed by measurements of the cGMP level. During normoxia, treatment with VEGF (5 ng/ml) increased permeability as well as cGMP content in the presence of several antioxidants. These results suggest that hypoxia-induced permeability in vitro is mediated by the VEGF/VEGF receptor system in an autocrine manner and is essentially dependent on reducing conditions stabilizing the second messenger NO as the mediator of changes in barrier function of BMEC.In this study, an in vitro model of the blood-brain barrier, consisting of porcine brain-derived microvascular endothelial cells (BMEC), was used to evaluate the mechanism of hypoxia-induced hyperpermeability. We show that hypoxia-induced permeability in BMEC was completely abolished by a neutralizing antibody to vascular endothelial growth factor (VEGF). In contrast, under normoxic conditions, addition of VEGF up to 100 ng/ml did not alter monolayer barrier function. Treatment with either hypoxia or VEGF under normoxic conditions induced a twofold increase in VEGF binding sites and VEGF receptor 1 (Flt-1) mRNA expression in BMEC. Hypoxia-induced permeability also was prevented by the nitric oxide (NO) synthase inhibitor N G-monomethyl-l-arginine, suggesting that NO is involved in hypoxia-induced permeability changes, which was confirmed by measurements of the cGMP level. During normoxia, treatment with VEGF (5 ng/ml) increased permeability as well as cGMP content in the presence of several antioxidants. These results suggest that hypoxia-induced permeability in vitro is mediated by the VEGF/VEGF receptor system in an autocrine manner and is essentially dependent on reducing conditions stabilizing the second messenger NO as the mediator of changes in barrier function of BMEC.

VEGF induces hyperpermeability by a direct action on endothelial cells

Vascular endothelial growth factor (VEGF) is a key regulator of vasculo- and angiogenesis. Earlier studies demonstrated a permeability-increasing effect of VEGF in skin tests, leading to its other name, vascular permeability factor. We wondered whether VEGF-induced hyperpermeability was a direct effect of VEGF on endothelial cells and studied the permeability of human and porcine endothelial cell monolayers in a well-characterized in vitro system. VEGF increased the hydraulic conductivity up to 20-fold and simultaneously decreased the albumin reflection coefficient. This effect occurred after a delay of 150 min, although VEGF-induced early endothelial cell activation was verified by enhanced inositol phosphate accumulation within 5 min and increased P-selectin expression within 15 min. Platelet-derived growth factor and granulocyte-macrophage colony-stimulating factor, two endothelial cell nonspecific mitogens, also stimulated phosphatidylinositol metabolism and P-selectin expression; however, they had no effect on endothelial permeability. The increase in intracellular cyclic nucleotide levels of human endothelial monolayers abolished VEGF-induced endothelial hyperpermeability. In summary, VEGF increased endothelial permeability by a direct action on endothelial cells. Based on the pattern of endothelial cell activation by growth factors, VEGF appears to be a unique stimulus.Vascular endothelial growth factor (VEGF) is a key regulator of vasculo- and angiogenesis. Earlier studies demonstrated a permeability-increasing effect of VEGF in skin tests, leading to its other name, vascular permeability factor. We wondered whether VEGF-induced hyperpermeability was a direct effect of VEGF on endothelial cells and studied the permeability of human and porcine endothelial cell monolayers in a well-characterized in vitro system. VEGF increased the hydraulic conductivity up to 20-fold and simultaneously decreased the albumin reflection coefficient. This effect occurred after a delay of 150 min, although VEGF-induced early endothelial cell activation was verified by enhanced inositol phosphate accumulation within 5 min and increased P-selectin expression within 15 min. Platelet-derived growth factor and granulocyte-macrophage colony-stimulating factor, two endothelial cell nonspecific mitogens, also stimulated phosphatidylinositol metabolism and P-selectin expression; however, they had no effect on endothelial permeability. The increase in intracellular cyclic nucleotide levels of human endothelial monolayers abolished VEGF-induced endothelial hyperpermeability. In summary, VEGF increased endothelial permeability by a direct action on endothelial cells. Based on the pattern of endothelial cell activation by growth factors, VEGF appears to be a unique stimulus.

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.

Vascular endothelial growth factor (VEGF) stimulates monocyte migration through endothelial monolayers via increased integrin expression

Monocytes play an important role in collateral vessel formation (arteriogenesis) by attaching to activated endothelium and by invading the walls of innate collateral vessels where they produce growth factors. Previous studies have demonstrated that this process can be promoted by several chemokines and growth factors. In this study we examined the interaction between monocytes and endothelium under stimulation of the angiogenic agent vascular endothelial growth factor (VEGF). We report here the novel finding that VEGF stimulates the expression of the alphaL-, alphaM- and beta2-integrin monomers. In functional assays and by using neutralizing antibodies it was shown that VEGF stimulates adhesion of monocytes to human umbilical vein endothelial cells (HUVEC), and increased transmigration through endothelial monolayers is dependent on interaction of monocyte beta2-integrins with its endothelial counter ligand ICAM-1. Based on these in vitro data we hypothesize that the positive effect of VEGF on arteriogenesis may involve monocyte activation.