Silvia Fischer

The Range of Adaptation by Collateral Vessels After Femoral Artery Occlusion

Natural adaptation to femoral artery occlusion in animals by collateral artery growth restores only ≈35% of adenosine-recruitable maximal conductance (Cmax) 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. Cmax reached 100% already at day 7 and had, after 4 weeks, surpassed (2-fold) the Cmax 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.

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.

In vitro effects of dexamethasone on hypoxia-induced hyperpermeability and expression of vascular endothelial growth factor.

Clinically, dexamethasone is known to reduce cerebral edema. To further investigate the mechanism of this neuroprotection, an in vitro model of brain-derived microvessel endothelial cells (BME cells) was used to investigate the effect of dexamethasone on hypoxia-induced hyperpermeability. Furthermore, the expression of vascular endothelial growth factor (VEGF), which is known to be the mediator of hypoxia-induced hyperpermeability, was evaluated. Dexamethasone (40 microg/ml=100 microM) decreased hypoxia-induced permeability and VEGF expression significantly during time periods of more than 3 h. The time dependence of the dexamethasone effect correlated with a changed mechanism by which hypoxia induced VEGF expression. This was deduced because hypoxia-induced hyperpermeability and VEGF mRNA level were decreased in the presence of an antisense oligonucleotide coding for a region which binds a mRNA stabilizing protein, but only up to 3 h of hypoxia. Furthermore, during this time period the half-life of VEGF mRNA was increased. Results suggest that dexamethasone only decreases transcriptional-induced VEGF expression and that this may be related to the efficacy of dexamethasone to treat brain edema.

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 Cγ (PLCγ), 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 PLCγ, 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 PLCγ and PI3‐K/AKT pathway leading to the activation of PKG. J. Cell. Physiol. 198: 359–369, 2004© 2003 Wiley‐Liss, Inc.

The functional role of blood platelet components in angiogenesis

The process of neovascularization greatly depends on the induction of the angiogenic phenotype of endothelial cells that is strictly controlled by humoral factors as well as by cellular communications in the vascular system. Although blood platelets contain several secretable pro- and antiangiogenic components, their overall role in angiogenesis remains poorly understood. In a mouse model of hypoxia-induced retinal angiogenesis, the situation of thrombocytopenia as well as inhibition of platelet aggregation by a highly specific alphaIIbbeta3-integrin antagonist or acetyl salicylic acid (Aspirin) administration, respectively, resulted in about 35-50% reduction of retinal neovascularization, compatible with a significant contribution of blood platelets in angiogenesis. Platelet remnants and microvesicles were found at sites of angiogenic sprouts. In vitro isolated platelets incorporated in a fibrin gel induced capillary sprouting of microvascular endothelial cells. Similarly, platelet releasate elevated the permeability of confluent endothelial cell monolayers to the same extent as hypoxia did. Platelet-derived VEGF as well as butanol-extractable lipid mediators were identified as predominant activators of angiogenesis, particularly of microvascular endothelial cell proliferation and migration. In addition, a synergistic effect between platelet-derived VEGF and bFGF in capillary sprouting and endothelial cell proliferation was found. Based on this proangiogenic role of platelets in neovascularization, anti-platelet substances can be considered as potent inhibitors of angiogenesis.

Complex formation with nucleic acids and aptamers alters the antigenic properties of platelet factor 4

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.

Flt-1, but not Flk-1 mediates hyperpermeability through activation of the PI3-K/Akt pathway.

Vascular endothelial growth factor (VEGF), a potent mediator of endothelial proliferation and migration, has an important role also in brain edema formation during hypoxia and ischemia. VEGF binds to the tyrosine kinase receptors Flt‐1 and Flk‐1. Yet, their relative importance for hypoxia‐induced hyperpermeability is not well understood. We used an in vitro blood‐brain barrier (BBB) model consisting of porcine brain microvascular endothelial cells (BMEC) to determine the role of Flt‐1 in VEGF‐induced endothelial cell (EC) barrier dysfunction. Soluble Flt‐1 abolished hypoxia/VEGF‐induced hyperpermeability. Furthermore, selective antisense oligonucleotides to Flt‐1, but not to Flk‐1, inhibited hypoxia‐induced permeability changes. Consistent with these data, addition of the receptor‐specific homolog placenta‐derived growth factor, which binds Flt‐1 but not Flk‐1, increased endothelial permeability to the same extent as VEGF, whereas adding VEGF‐E, a viral VEGF molecule from the orf virus family activating Flk‐1 and neuropilin‐1, but not Flt‐1, did not show any effect. Using the carcinoma submandibular gland cell line (CSG), only expressing Flt‐1, it was demonstrated that activation of Flt‐1 is sufficient to induce hyperpermeability by hypoxia and VEGF. Hyperpermeability, induced by hypoxia/VEGF, depends on activation of phosphatidylinositol 3‐kinase/Akt (PI3‐K/Akt), nitric oxide synthase (NOS) and protein kinase G (PKG). The activation of the PI3‐K/Akt pathway by hypoxia was confirmed using an in vivo mice hypoxia model. These results demonstrate that hypoxia/VEGF‐induced hyperpermeability can be mediated by activation of Flt‐1 independently on the presence of Flk‐1 and indicate a central role for activation of the PI3‐K/Akt pathway, followed by induction of NOS and PKG activity. J. Cell. Physiol. 212: 236–243, 2007.

Signaling mechanism of extracellular RNA in endothelial cells

Extracellular RNA has been shown to induce vascular endothelial growth factor (VEGF)‐dependent hyperpermeability in vivo as well as in vitro. Studies were performed to investigate the mechanism of these effects. For permeability studies primary cultures of porcine brain‐derived microvascular endothelial cells (BMECs) and for all other analytical studies the human brain endothelial cell line HCMEC/D3 or human umbilical vein endothelial cells (HUVECs) were used. RNA, but not DNA, initiated signaling events by binding of VEGF to neuropilin‐1, followed by VEGF‐R2 phosphorylation, activation of phospholipase C (PLC), and intracellular release of Ca2+. Activation of these pathways by RNA also resulted in the release of von Willebrand Factor from Weibel‐Palade bodies. Pretreat‐ment of cells with heparinase totally abrogated the RNA‐induced permeability changes, whereas RNA together with VEGF completely restored VEGF‐R2‐mediated hyperpermeability. Although poly:IC increased the interleukin‐6 release via activation of toll‐like receptor‐3 (TLR‐3), permeability changes mediated by poly:IC or RNA remained unchanged after blocking TLR‐3 or NF‐kB activation. These results indicate that extracellular RNA serves an important cofactor function to engage VEGF for VEGF‐R2‐dependent signal transduction, reminiscent of the coreceptor mechanism mediated by proteoglycans, which might be of relevance for the mobilization and cellular activities of RNA‐binding cytokines in general.—Fischer, S.,Nishio, M., Peters, S. C., Tschernatsch, M., Walberer, M., Weidemann, S., Heidenreich, R., Couraud, P. O., Weksler, B. B., Romero, I. A., Gerriets, T., Preissner, K. T. Signaling mechanism of extracellular RNA in endothelial cells. FASEBJ. 23, 2100–2109 (2007)

Expression and localisation of vascular ribonucleases in endothelial cells.

The functions of extracellular RNA in the vascular system as new procoagulatory and permeability-increasing factor in vivo and in vitro were shown to be counteracted by pancreatic type RNase1. Based on the identification of RNase1 in plasma and serum, it is proposed that the enzyme is expressed by vascular cells to contribute in the regulation of extracellular RNA. It is demonstrated that RNase1 and RNase5 (also termed angiogenin) were differentially expressed in various types of endothelial cells, whereby human umbilical vein endothelial cells (HUVEC) expressed and released the highest concentration of active RNase1. Expression and release of RNase5 were similar in all types of endothelial cells tested. Both RNases were constitutively produced and secreted, whereby a portion of RNase1, but not RNase5, was stored in Weibel-Palade bodies, co-localising with von Willlebrand factor and P-selectin. Accordingly, immediate release of RNase1 from these granules was demonstrated in vitro and in vivo using Weibel-Palade body exocytosis-inducing agents. Additionally, extracellular RNA or poly:IC (but not DNA) induced this short-term release of RNase1. Our results indicate that vascular RNase1 and RNase5 are mainly produced by vascular endothelial cells and can serve, depending on the vascular bed, different functions in vascular homeostasis and endothelial cell responses.

Extracellular RNA promotes leukocyte recruitment in the vascular system by mobilising proinflammatory cytokines

Extracellular RNA (eRNA), released from cells under conditions of injury or vascular disease, acts as potent prothrombotic factor and promotes vascular hyperpermeability related to oedema formation in vivo. In this study, we aimed to investigate the mechanism by which eRNA triggers inflammatory processes, particularly associated with different steps of leukocyte recruitment. Using intravital microscopy of murine cremaster muscle venules, eRNA (but not DNA) significantly induced leukocyte adhesion and transmigration in vivo, which was comparable in its effects to the function of tumour-necrosis-factor-α (TNF-α). In vitro, eRNA promoted adhesion and transmigration of monocytic cells on and across endothelial cell monolayers. eRNA-induced monocyte adhesion in vitro was mediated by activation of the vascular endothelial growth factor (VEGF)/VEGF-receptor-2 system and was abolished by neutralising antibodies against intercellular adhesion molecule-1 or the β2-integrin Mac-1. Additionally, eRNA induced the release of TNF-α from monocytic cells in a time- and concentration-dependent manner, which involved activation of TNF-α-converting enzyme (TACE) as well as the nuclear factor κB signalling machinery. In vivo, inhibiton of TACE significantly reduced eRNA-induced leukocyte adhesion. Our findings present evidence that eRNA in connection with tissue/vascular damage provokes a potent inflammatory response by inducing leukocyte recruitment and by mobilising proinflammatory cytokines from monocytes.