Formation of cerebral oedema caused by vascular leakage is a major problem in various injuries of the CNS, such as stroke, head injury and high-altitude illness. A common feature of all these disorders is the fact that they are associated with tissue hypoxia. Hypoxia has therefore been suggested to be an important pathogenic factor for the induction of vascular leakage in the brain. Vascular endothelial growth factor (VEGF) is known as the major inducer of angiogenesis. Originally, however, it was described as a vascular permeability factor. As VEGF gene expression was shown to be upregulated by hypoxia, increased VEGF expression may link hypoxia and vascular leakage in the CNS in vivo. To delineate the role of VEGF in vascular leakage in the brain, we studied the effect of hypoxia on VEGF expression and vascular permeability in the brains of mice in vivo. Hypoxic exposure led to a significant increase in the levels of VEGF mRNA and protein in mouse brain that correlated with the severity of the hypoxic stimulus. Measurement of vascular permeability using the fluorescent marker sodium fluorescein revealed a two-fold increase in fluorescence intensity in hypoxic brains, indicative of significant vascular leakage. Inhibition of VEGF activity by a neutralizing antibody completely blocked the hypoxia-induced increase in vascular permeability. In conclusion, our data show that VEGF is responsible for hypoxia-induced augmentation in vascular leakage following tissue hypoxia. Our findings might provide the basis for new therapeutic concepts for the treatment of cerebral oedema.

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Abstract-Natural adaptation to femoral artery occlusion in animals by collateral artery growth restores only Ϸ35% of adenosine-recruitable maximal conductance (C max ) probably because initially elevated fluid shear stress (FSS) quickly normalizes. We tested the hypothesis whether this deficit can be mended by artificially increasing FSS or whether anatomical restraints prevent complete restitution. We chronically increased FSS by draining the collateral flow directly into the venous system by a side-to-side anastomosis between the distal stump of the occluded femoral artery and the accompanying vein. After reclosure of the shunt collateral flow was measured at maximal vasodilatation. C max reached 100% already at day 7 and had, after 4 weeks, surpassed (2-fold) the C max of the normal vasculature before occlusion. Expression profiling showed upregulation of members of the Rho-pathway (RhoA, cofilin, focal adhesion kinase, vimentin) and the Rho-antagonist Fasudil markedly inhibited arteriogenesis. The activities of Ras and ERK-1,-2 were markedly increased in collateral vessels of the shunt experiment, and infusions of L-NAME and L-NNA strongly inhibited MAPK activity as well as shunt-induced arteriogenesis. Infusions of the peroxinitrite donor Sin-1 inhibited arteriogenesis. The radical scavengers urate, ebselen, SOD, and catalase had no effect. We conclude that increased FSS can overcome the anatomical restrictions of collateral arteries and is potentially able to completely restore maximal collateral conductance. Increased FSS activates the Ras-ERK-, the Rho-, and the NO-(but not the Akt-) pathway enabling collateral artery growth. Key Words: arteriogenesis Ⅲ fluid shear stress Ⅲ shunt Ⅲ growth factors Ⅲ microarrays T he restoration of maximal conductance (C max ) in animals after arterial occlusion remains defective (35% in the canine coronary circulation 1 and 40% in the rabbit hind limb 2 ) in spite of the fact that normal resting blood flow is reached early. As a consequence exercise testing in experimental animals reveals defects similar to those in human patients. 3 It was not known until now whether collateral vessels are potentially able to restore the full dilatory reserve of a normal vascular bed. Many observations would predict that this is not the case: the multitude of small vessels that replace an occluded artery is inefficient according to Poissieulle's Law, and the tortuosity of collateral vessels offers finite resistance because of curvature flow and increased collateral length. 4 One reason for the defective adaptation may lie in the fact that fluid shear stress normalizes prematurely: FSS falls by the third power of the growing radius. We tested the hypothesis whether a sustained increase of FSS is able to prolong the growth process and to restore normal maximal conductance. The method to achieve this was the creation of a shunt between the distal stump of the occluded femoral artery and the accompanying vein. 5 The novelty of the present findings is the demonstration that C max can be reach...

Cell injury leads to exposure of intracellular material and is associated with increased permeability of vessels in the vicinity of the damage. Here, we demonstrate that natural extracellular RNA as well as artificial RNA (poly- I IntroductionBrain homeostasis is maintained by the blood-brain barrier (BBB), which forms a mechanical and functional threshold between the central nervous system and the systemic circulation. The barrier is relatively impermeable to ions, many amino acids, small peptides, and proteins, and thus contributes to the maintenance of a specific neural tissue environment. In vertebrates, the BBB exists at the level of the endothelial cells that form brain capillaries 1 in order to regulate and limit the degree of trans-and paracellular flux. 2 The tight barrier properties of the BBB result from the absence of fenestrations, the low number of pinocytotic vesicles, and the presence of tight intercellular junctions between endothelial cells with extremely high electrical resistance. 3 Pathologic conditions associated with brain tumors, head injury, or ischemic stroke are accompanied by endothelial-cell dysfunction, leading to increased permeability across the BBB, which might lead to the development of vasogenic cerebral edema. 4,5 Vascular endothelial growth factor (VEGF) as a hypoxia/ischemia inducible protein in vitro and in vivo is one of the strongest natural permeability factors 6 and a likely candidate for the development of ischemia-and tumor-induced vasogenic brain edema. 7-9 VEGF stimulates endothelial-cell growth and migration in vitro 10,11 and angiogenesis in vivo. 6,12 VEGF was originally described as a potent vascular permeability factor responsible for the accumulation of plasma protein-rich fluid in the ascites of patients with tumors. 13 Structurally, VEGF exists as a dimeric glycoprotein of molecular weight (Mr) 34 000 to 42 000 and is related to the platelet-derived growth factor family of molecules. 14 Although VEGF is the product of a single gene, 6 differentially spliced isoforms between 121 and 206 amino acid residues exist in humans 15,16 that exhibit similar functional activities. Different isoforms are distinguished by their affinity for heparin: although VEGF 121 does not bind heparin, VEGF 165 has moderate affininity for heparin, whereas VEGF 189 and VEGF 206 bind heparin with high affinity. 17 VEGF exerts its multiple actions by ligation with tyrosine kinase receptors, VEGFreceptor 1 (VEGF-R1), as well as VEGF-R2, [18][19][20] which are expressed on vascular endothelial cells. A third member, VEGF-R3 is expressed on lymphatic endothelial cells. 21 During pathologic conditions of the brain associated with tumor burden, stroke, or head injury, nucleic acids might be released by damaged cells. RNA-proteolipid complexes were detected in the circulation of patients with cancer and were suggested to represent a specific secretory product of cancer cells. 22 Accordingly, circulating RNA is present in blood plasma of patients with tumors. 23 The presence of specific types ...

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.

Key Points• PF4 binds to nucleic acids and thereby exposes the epitope to which anti-PF4/ heparin antibodies bind.• PF4/aptamer complexes can induce an immune response resembling heparin-induced thrombocytopenia.The tight electrostatic binding of the chemokine platelet factor 4 (PF4) to polyanions induces heparin-induced thrombocytopenia, a prothrombotic adverse drug reaction caused by immunoglobulin G directed against PF4/polyanion complexes. This study demonstrates that nucleic acids, including aptamers, also bind to PF4 and enhance PF4 binding to platelets. Systematic assessment of RNA and DNA constructs, as well as 4 aptamers of different lengths and secondary structures, revealed that increasing length and double-stranded segments of nucleic acids augment complex formation with PF4, while single nucleotides or single-stranded polyA or polyC constructs do not. Aptamers were shown by circular dichroism spectroscopy to induce structural changes in PF4 that resemble those induced by heparin. Moreover, heparin-induced anti-human-PF4/ heparin antibodies cross-reacted with human PF4/nucleic acid and PF4/aptamer complexes, as shown by an enzyme immunoassay and a functional platelet activation assay. Finally, administration of PF4/44mer-DNA protein C aptamer complexes in mice induced anti-PF4/aptamer antibodies, which cross-reacted with murine PF4/heparin complexes. These data indicate that the formation of anti-PF4/heparin antibodies in postoperative patients may be augmented by PF4/nucleic acid complexes. Moreover, administration of therapeutic aptamers has the potential to induce anti-PF4/polyanion antibodies and a prothrombotic diathesis. (Blood. 2013;122(2):272-281) IntroductionThe chemokine platelet factor 4 (PF4) is released from platelet a-granules during platelet activation 1 and binds, due to its high positive charge, to many negatively charged polyanions, including heparin. PF4 forms large multimolecular complexes with heparin that are highly immunogenic. 2,3 The resulting immunoglobulin G (IgG) antibodies are the cause of heparin-induced thrombocytopenia (HIT), a prothrombotic adverse drug effect. 4 In HIT, multimolecular complexes composed of PF4, heparin, and anti-PF4/heparin IgG cross-link platelet FcgIIa receptors, 5 triggering platelet activation, microparticle formation, and thrombin generation, with ;50% of affected patients developing thrombosis. 6 Recently, we showed that PF4 binds to polyanions on the surface of bacteria, thereby forming multimolecular complexes that are recognized by human anti-PF4/heparin antibodies. More specifically, we identified the PF4 binding site on gram-negative bacteria as the phosphate groups of lipid A. 7 Based on this observation, we hypothesized that nucleic acids might also form multimolecular complexes with PF4 because they also expose multiple negatively charged phosphate groups. This idea was fostered by previous findings that salmon sperm DNA could substitute heparin in HIT-IgG-induced platelet activation. 8Plasma levels of extracellular nucleic acids are re...

Background -Despite optimal therapy, the morbidity and mortality of patients presenting

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