VEGF-induced Permeability Research Articles

A Vascular Endothelial Growth Factor High Affinity Receptor 1-specific Peptide with Antiangiogenic Activity Identified Using a Phage Display Peptide Library

Vascular endothelial growth factor (VEGF) is known to play a predominant role in tumor angiogenesis and metastasis formation that is mediated by its interactions with two tyrosine kinase receptors, VEGFRI (Flt-1) and VEGFRII (KDR). Inhibition of VEGF-dependent events in tumor tissues is known to enhance apoptosis and to suppress tumor growth. A novel peptide, SP5.2, which selectively binds Flt-1 and inhibits a broad range of VEGF-mediated events, was identified using a phage-display library screening. The fluorescein-labeled SP5.2 specifically bound to VEGF-stimulated primary human cerebral endothelial cells (HCECs), whereas non-stimulated HCECs, as well as human neuroblastoma cells (ShyY) did not show any interaction with the peptide. SP5.2 prevented proliferation of cultured primary human umbilical vein endothelial cells induced by recombinant human VEGF165 with an IC50 of 5 microm. SP5.2 was also shown to antagonize VEGF- and PLGF-induced, but not basic fibroblast growth factor-induced proliferation of HCECs. In contrast to "scrambled" peptide, SP5.2 was also found to selectively inhibit VEGF-stimulated migration of HCECs. The in vitro analysis of antiangiogenic activity of SP5.2 using a capillary-like tube formation assay showed that VEGF-induced angiogenesis of HCECs grown on Matrigel was completely inhibited in the presence of 10 microm SP5.2. Further studies demonstrated that SP5.2 prevented VEGF-induced permeability increase in HCECs monolayers. To explore whether SP5.2 can be used as a targeting agent, chemical and recombinant conjugates of SP5.2 with reporter proteins (peroxidase and beta-galactosidase) were produced. The resulting products showed significant increases (200-fold for SP5.2-beta-gal and 400-fold for SP5.2-peroxidase) in binding affinity to recombinant Flt-1 compared with the original synthetic SP5.2, suggesting that conjugate with therapeutic activity in nanomolar range could potentially be developed based on SP5.2 structure.

Erythropoietin protects the in vitro blood-brain barrier against VEGF-induced permeability.

The blood-brain barrier (BBB) ensures the homeostasis of the brain microenvironment, mostly through complex tight junctions between brain endothelial cells that prevent the passage of hydrophilic molecules from blood to brain and vice versa. A recent study has shown in vivo that systemic administration of erythropoietin (Epo) protects against brain injury. Using an in vitro model of the bovine BBB, we observed that the expression of the Epo receptor is modulated by its ligand and hypoxic stimuli such as vascular endothelial growth factor (VEGF) treatment. In addition, Epo protects against the VEGF-induced permeability of the BBB, decreases the levels of endothelial nitric oxide synthase and restores junction proteins. The kinetic transport experiments revealed the capacity of Epo to cross the in vitro BBB in a saturable and specific way. Our results suggest a new mechanism for Epo-induced neuroprotection, in which circulating Epo controls and maintains the BBB through an Epo receptor signalling pathway and the re-establishment of cell junctions.

Simultaneous activation of several second messengers in hypoxia‐induced hyperpermeability of brain derived endothelial cells

In vivo, ischemia is known to damage the blood-brain barrier (BBB) leading to the development of vasogenic brain edema. Hypoxia-induced vascular endothelial growth factor (VEGF) has been shown to be a key regulator of these permeability changes. However, the signaling pathways that underlie VEGF-induced hyperpermeability are incompletely understood. In this study, we demonstrate that hypoxia- and VEGF-induced permeability changes depend on activation of phospholipase Cgamma (PLCgamma), phosphatidylinositol 3-kinase/Akt (PI3-K/Akt), and protein kinase G (PKG). Inhibition of mitogen-activated protein kinases (MAPK) and of the protein kinase C (PKC) did not affect permeability at all. Paralleling hypoxia- and VEGF-induced permeability changes, localization of the tight junction proteins occludin, zonula occludens-1 (ZO-1), and ZO-2 along the cell membrane changed from a continuous to a more discontinuous expression pattern during hypoxia. In particular, localization of ZO-1 and ZO-2 expression moved from the cell membrane to the cytoplasm and nucleus whereas occludin expression remained at the cell membrane. Inhibition of PLCgamma, PI3-kinase, and PKG abolished these hypoxia-induced changes. These findings demonstrate that hypoxia and VEGF induce permeability through rearrangement of endothelial junctional proteins which involves activation of the PLCgamma and PI3-K/AKT pathway leading to the activation of PKG.

Induction of vascular networks in adult organs: implications to proangiogenic therapy.

Most therapeutic proangiogenic approaches rely on the premise that a single factor (e.g., VEGF) can induce new vascular loops properly connected to the existing vasculature of an ill-perfused organ (e.g., the ischemic myocardium). To gain insights into VEGF-driven neovascularization in the adult, in general, and to uncover inherent obstacles in VEGF-based therapeutic angiogenesis, we have used a transgenic system for conditional "on" and "off" switching of VEGF expression in selected adult organs and analytical methods for three-dimensional visualization of the induced vascular network. VEGF induces massive accumulation of endothelial progenitor cells and their subsequent coalescence into new vascular loops. Continual ramification of the vasculature and fusion of adjacent vessels culminate in formation of hemangioma-like vessels. In the absence of guiding cues, abnormal branching patterns and random connections with the existing network are formed. Following cessation of the VEGF stimulus, the acquired network undergoes extensive hemodynamic remodeling acting to "normalize" its architecture. Premature withdrawal of VEGF leads to regression of most acquired vessels, thus challenging the utility of therapeutic approaches relying on short stimulus duration. Overproduction of VEGF leads also to highly disruptive edema, thereby emphasizing the need to uncouple VEGF-induced permeability from its angiogenic activity. This genetic switch system thus highlights remaining problems critical for the implementation of proangiogenic therapy.

Small GTP-binding protein Rac is an essential mediator of vascular endothelial growth factor-induced endothelial fenestrations and vascular permeability.

Vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) induces both angiogenesis and vascular permeability. Although its angiogenic activity has been well characterized, the signaling pathways of VEGF-induced permeability remain poorly understood. Using the mouse corneal micropocket assay, Miles assay, and a combination of cytochemical, electron microscopic, and biochemical assays, we demonstrate that VEGF-induced vascular leakage partly can be separated from its angiogenic activity. VEGF but not FGF-2 induced capillaries with a highly fenestrated endothelium, a feature linked with increased vascular permeability. A cell-permeable Rac antagonist (TAT-RacN17) converted VEGF-induced, leaky vascular plexuses into well-defined vascular networks. In addition, this Rac mutant blocked formation of VEGF-induced endothelial fenestrations and vascular permeability but only partially inhibited angiogenesis. Studies on endothelial cell cultures further revealed that VEGF stimulated phosphorylation of VEGF receptor-2 (VEGFR-2), leading to activation of Rac as well as increased phosphorylation of phospholipase Cgamma (PLCgamma), protein kinase B (Akt), endothelial nitric oxide synthase (eNOS), and extracellular regulated kinase (Erk1/2). We further found that phosphatidylinositol-3-OH kinase (PI3K) acted upstream of Rac and Akt-eNOS in VEGF/VEGFR-2 signaling. Our findings indicate that the small GTP-binding protein Rac is a key component in mediation of VEGF-induced vascular permeability but less so in neovascularization. This may have conceptual implications for applying Rac antagonists in treatment and prevention of VEGF-induced vascular leakage and edema in connection with ischemic disorders.

VEGF-induced paracellular permeability in cultured endothelial cells involves urokinase and its receptor.

Vascular endothelial growth factor/vascular permeability factor (VEGF) has been implicated in blood/tissue barrier dysfunctions associated with pathological angiogenesis, but the mechanisms of VEGF-induced permeability increase are poorly understood. Here, the role of VEGF-induced extracellular proteolytic activities on the endothelial cell permeability increase is evaluated. Confluent monolayers of bovine retinal microvascular endothelial (BRE) cells grown on porous membrane were treated with VEGF or urokinase plasminogen activator (uPA), and permeability changes were analyzed. uPA-induced permeability was rapid and sustained, but VEGF-induced permeability showed a biphasic pattern: a rapid and transient phase (1-2 h) followed by delayed and sustained phase (6-24 h). The delayed, but not the early phase of VEGF-induced permeability, was blocked by anti-uPA or anti-uPAR (uPA receptor) antibodies and was accompanied by reduced transendothelial electrical resistance, indicating the paracellular route of permeability. Confocal microscopy and Western blotting showed that VEGF treatment increased free cytosolic beta-catenin, which was followed by beta-catenin nuclear translocation, upregulation of uPAR, and downregulation of occludin. Membrane-bound occludin was released immediately after uPA treatment, but with a long delay after VEGF treatment, suggesting a requirement for uPAR gene expression. In conclusion, VEGF induces a sustained paracellular permeability in capillary endothelial cells that is mediated by activation of the uPA/uPAR system.

VEGF Increases Permeability of the Endothelial Cell Monolayer by Activation of PKB/akt, Endothelial Nitric-Oxide Synthase, and MAP Kinase Pathways

VEGF is a key regulator of vascular permeability. However, its signaling pathways are incompletely understood. We tested the hypothesis that VEGF regulates endothelial cell (EC) permeability by activating PKB/akt, NOS, and MAP kinase dependent pathways using human umbilical vein EC (HUVEC). Permeability was measured from FITC–dextran 70-kDa flux across the EC monolayer at baseline and after VEGF at 0.034, 0.068, 1, 10, and 100 nM. VEGF increased HUVEC permeability to FITC–dextran in a dose-dependent manner. VEGF (1 nM) increased permeability from 3.9 × 10−6 ± 0.7 × 10−6 to 14.0 × 10−6 ± 1.7 × 10−6 cm/s (mean ± SEM; P < 0.001). Permeability changes were also assessed after treatment with 1, 10, and 100 nM wortmannin (PI 3-kinase inhibitor); 0.01, 0.1, and 1.0 nM LY294002 (PI 3-kinase inhibitor); 200 μM l-NMMA (NOS inhibitor); 2.7 μM AG126 (p42/44MAPK inhibitor); and 0.006, 0.06, and 0.6 μM SB203580 (p38MAPK inhibitor). All inhibitors blocked VEGF-induced permeability changes. Our data demonstrate that (1) VEGF increases permeability of EC monolayers in a dose-dependent fashion, and (2) VEGF-induced permeability is mediated through PI-3 kinase-PKB, NOS, and MAP-kinase signaling cascades. These observations suggest that microvascular hyperpermeability associated with inflammation and vascular disease is mediated by activation of these EC signaling pathways.

A permissive role for tumor necrosis factor in vascular endothelial growth factor–induced vascular permeability

Vascular endothelial growth factor (VEGF) induces both angiogenesis and an increase in vascular permeability, 2 processes that are considered to be important for both tumor growth and the delivery of drugs to the site of tumors. This study demonstrates that transmembrane expression of tumor necrosis factor (tmTNF) is up-regulated in the endothelium of a murine methylcholanthrene (meth A)-induced sarcoma in comparison to the adjacent normal dermal vasculature and is also present on cultivated human endothelial cells. It is further shown that tmTNF is required for VEGF-mediated endothelial hyperpermeability in vitro and in vivo. This permissive activity of TNF appears to be selective, because anti-TNF antibodies ablated the VEGF-induced permeability but not proliferation of cultivated human endothelial cells. Furthermore, tnf gene-deficient mice show no obvious defects in vascularization and develop normally but failed to respond to administration of VEGF with an increase in vascular permeability. Subsequent studies indicated that the tmTNF and VEGF signaling pathways converge at the level of a secondary messenger, the "stress-activated protein kinase-2" (SAPK-2)/p38: (1) up-regulated endothelial expression of tmTNF resulted in the continuous activation of SAPK-2/p38 in vitro, and (2) an inhibitor of SAPK-2/p38 activation abolished the vascular permeability activity of VEGF in vivo. In conclusion, the study's finding that continuous autocrine signaling by tmTNF sensitizes endothelial cells to respond to VEGF by increasing their vascular permeability provides new therapeutic concepts for manipulating vascular hyperpermeability.

Barbiturates decrease the expression of vascular endothelial growth factor in hypoxic cultures of porcine brain derived microvascular endothelial cells

Vascular endothelial growth factor (VEGF) is known to be produced in higher amounts during hypoxia by a variety of cell types and has been shown to increase the permeability of brain derived microvascular endothelial cells (BMEC) during hypoxia by an autocrine mechanism. Because the barbiturates, methohexital (MH) and thiopental (TP), induced a dose-dependent reduction in hypoxia-induced permeability changes of BMEC, the effect of both barbiturates on the VEGF expression during hypoxia was investigated. Both barbiturates decreased the hypoxia-induced expression of VEGF in BMEC in a concentration-dependent manner. This effect is partly caused by the impairment of the hypoxia-induced VEGF mRNA stabilization. VEGF-induced permeability changes during normoxia were unaffected by the barbiturates suggesting that MH and TP are directly reducing hypoxia-induced VEGF synthesis. In conclusion, the inhibiting effect of these barbiturates on the hypoxia-induced VEGF expression results in the decreased permeability of the BMEC monolayer during hypoxia, which may contribute to the described neuroprotective action of barbiturates by reduction of brain edema formation.

Vascular endothelial growth factor-induced retinal permeability is mediated by protein kinase C in vivo and suppressed by an orally effective beta-isoform-selective inhibitor.

Increased vascular permeability and excessive neovascularization are the hallmarks of endothelial dysfunction, which can lead to diabetic macular edema and proliferative diabetic retinopathy in the eye. Vascular endothelial growth factor (VEGF) is an important mediator of ocular neovascularization and a known vasopermeability factor in nonocular tissues. In these studies, we demonstrate that intravitreal injection of VEGF rapidly activates protein kinase C (PKC) in the retina at concentrations observed clinically, inducing membrane translocation of PKC isoforms alpha, betaII, and delta and >threefold increases in retinal vasopermeability in vivo. The effect of VEGF on retinal vascular permeability appears to be mediated predominantly by the beta-isoform of PKC with >95% inhibition of VEGF-induced permeability by intravitreal or oral administration of a PKC beta-isoform-selective inhibitor that did not inhibit histamine-mediated effects. These studies represent the first direct demonstration that VEGF can increase intraocular vascular permeability through activation of PKC in vivo and suggest that oral pharmacological therapies involving PKC beta-isoform-selective inhibitors may prove efficacious for the treatment of VEGF-associated ocular disorders such as diabetic retinopathy.

Vascular endothelial growth factor affects permeability of brain microvessel endothelial cells in vitro.

Vascular endothelial growth factor (VEGF), which stimulates endothelial cell growth and induces hyperpermeability of the microvasculature, plays an important role in normal and tumor-vasculature development and tumor edema generation. In this study, we investigated the effect of VEGF on the permeability of cultured bovine brain microvessel endothelial cells (BMECs), an in vitro blood-brain barrier (BBB) model. We found that addition of purified VEGF to both the apical and basolateral sides of the BMEC monolayers increased the permeability of the monolayer to [14C]sucrose (approximately 3-fold). A more significant increase in permeability was observed when VEGF was applied to the basolateral side of the monolayer (3-fold) than to the apical side (1.5-fold). The permeability-increasing activity of VEGF on the BMEC monolayers is both dose and time dependent. The VEGF-induced permeability increase in BMECs requires a long incubation time with VEGF, and the effect is durable. These results suggest that this cell culture system may be useful for exploring the role of VEGF in regulating the permeability of the BBB, for studying the mechanism of the permeability-increasing effect of VEGF on the endothelial cells, and for evaluating the strategies to regulate the activity of VEGF.