The effect of circulating iron on barrier integrity of primary human endothelial cells.

Iron is hypothesized to be one of the contributors to cardiovascular disease and its levels in the circulation may correlate with cardiovascular risk. The aim of this study is to investigate the mechanisms that underlie the effects of iron on the barrier function of primary human endothelium. We used Human Umbilical Vein Endothelial Cells (HUVEC) to investigate the effects of Fe3+ using electric cell-substrate impedance sensing, microscopy, western blot and immunofluorescence microscopy. Exposure to Fe3+ caused EC elongation and upregulation of stress-induced proteins. Analysis of barrier function showed a dose-dependent drop in endothelial integrity, which was accompanied by Reactive Oxygen Species (ROS) production and could partly be prevented by ROS scavengers. Inhibition of contractility by the ROCK inhibitor Y27632, showed even more effective rescue of barrier integrity. Using western blot, we detected an increase in expression of the small GTPase RhoB, an inducer of EC contraction, and a small decrease in VE-cadherin, suggestive for an iron-induced stress response. Co-stimulation by TNFα and iron, used to investigate the role of low-grade inflammation, revealed an additive, negative effect on barrier integrity, concomitant with an upregulation of pro-inflammatory markers ICAM-1 and RhoB. Iron induces a response in HUVEC that leads to endothelial activation and a pro-inflammatory state measured by loss of barrier integrity which can be reversed by ROS scavengers, combined with inhibition of contractility. These data suggest that ROS-mediated damage of the vascular endothelium could contribute to the increased cardiovascular risk which is associated with elevated levels of circulating iron.

Regulated membrane localization of Tiam1, mediated by the NH2-terminal pleckstrin homology domain, is required for Rac-dependent membrane ruffling and C-Jun NH2-terminal kinase activation.

Rho-like GTPases, including Cdc42, Rac, and Rho, regulate signaling pathways that control actin cytoskeletal structures and transcriptional activation. The Tiam1 gene encodes an activator of Rac1, and similarly to constitutively activated (V12)Rac1, overexpression of Tiam1 in fibroblasts induces the formation of membrane ruffles. Tiam1 contains a Dbl homology (DH) domain and adjacent pleckstrin homology (PH) domain, hallmarks for activators of Rho-like GTPases. Unique for Tiam1 are an additional PH domain and a Discs-large homology region in the NH2-terminal part of the protein. Here we show that both in fibroblasts and COS cells, membrane localization of Tiam1 is required for the induction of membrane ruffling. A detailed mutational analysis, in combination with confocal laser scanning microscopy and immunoelectron microscopy, demonstrates that the NH2-terminal PH domain of Tiam1, but not the DH-adjacent PH domain, is essential for membrane association. This NH2-terminal PH domain of Tiam1 can be functionally replaced by the myristoylated membrane localization domain of c-Src, indicating that the primary function of this PH domain is to localize the protein at the membrane. After serum starvation, both membrane association of Tiam1 and ruffling can be induced by serum, suggesting that receptor stimulation induces membrane translocation of Tiam1. Similar to V12Rac1, Tiam1 stimulates the activity of the c-Jun NH2-terminal kinase (JNK). This Rac-dependent stimulation of JNK also requires membrane association of Tiam1. We conclude that the regulated membrane localization of Tiam1 through its NH2-terminal PH domain determines the activation of distinct Rac-mediated signaling pathways.

Inhibition of invasion of epithelial cells by Tiam1-Rac signaling.

Tiam1 encodes an exchange factor for the Rho-like guanosine triphosphatase Rac. Both Tiam1 and activated RacV12 promote invasiveness of T lymphoma cells. In epithelial Madin-Darby canine kidney (MDCK) cells, Tiam1 localized to adherens junctions. Ectopic expression of Tiam1 or RacV12 inhibited hepatocyte growth factor-induced scattering by increasing E-cadherin-mediated cell-cell adhesion accompanied by actin polymerization at cell-cell contacts. In Ras-transformed MDCK cells, expression of Tiam1 or RacV12 restored E-cadherin-mediated adhesion, resulting in phenotypic reversion and loss of invasiveness. These data suggest an invasion-suppressor role for Tiam1 and Rac in epithelial cells.

Endothelial signalling by Ig-like cell adhesion molecules.

Leukocyte transendothelial migration is controlled by chemokine-induced signalling in leukocytes and integrin-ligand-induced bidirectional signalling in both leukocytes and endothelial cells. It is now generally accepted that endothelial signalling following leukocyte adhesion, serves to facilitate the crossing of the endothelium, be it via the paracellular or transcellular route. This brief overview discusses the main findings within this area and highlights some recent findings that shed new light on adhesion-induced signalling in the context of leukocyte transendothelial migration.

Endothelial dysfunction as a long-term effect of late onset hypertensive pregnancy disorders: High BMI is key.

OBJECTIVE: Hypertensive disorders during pregnancy increase cardiovascular risk later in life by 2 to 9-fold. Endothelial activation is one of the underlying mechanisms of cardiovascular risk. Therefore, we decided to investigate endothelial activation in primiparous women, 2.5 years after a hypertensive pregnancy disorder. STUDY DESIGN: Plasma samples were taken from women 2.5 years after gestational hypertension (GH) or late onset preeclampsia (cases) and from women 2.5 years after a normotensive pregnancy (controls). We studied the effects of patient plasma on the endothelial barrier function of primary human umbilical vein endothelial cells (HUVECs) using Electric Cell-Substrate Impedance Sensing (ECIS) and we measured levels of endothelial activation markers soluble intercellular adhesion molecule 1 (sICAM-1) and soluble endothelial selectin (sE-selectin) in the plasma samples of patients. RESULTS: Plasma from primiparous women with a history of late onset preeclampsia disrupted the endothelial barrier more than plasma from women with a history of GH. Endothelial resistance was reduced by 22% in samples taken after preeclampsia, 16% after normotensive pregnancy and 3% after GH (p ≤ 0.0001 GH versus preeclampsia and p = 0.0003 versus normotensive pregnancy). We did not find differences in the levels of soluble endothelial activation markers (sICAM-1 p = 0.326 and sE-selectin p = 0.978). However, the BMI ≥25 showed a strong correlation with increased levels of sICAM-1 (p = 0.046) and sE-selectin (p = 0.002). CONCLUSION: Our results indicate that GH and late onset preeclampsia are distinct disease entities with a different pathogenic mechanism underlying their cardiovascular risk. Furthermore, this study supports the hypothesis that these two diseases are early manifestations of cardiovascular vulnerability due to an unfavorable risk profile, and that obesity plays a main role. Our results suggest that this high-risk female population would be eligible for preventive care.

The minor histocompatibility antigen 1 (HMHA1)/ArhGAP45 is a RacGAP and a novel regulator of endothelial integrity.

Endothelial cells line the vasculature and act as gatekeepers that control the passage of plasma, macromolecules and cells from the circulation to the interstitial space. Dysfunction of the endothelial barrier can lead to uncontrolled leak or edema. Vascular leakage is a hallmark of a range of diseases and despite its large impact no specialized therapies are available to prevent or reduce it. RhoGTPases are known key regulators of cellular behavior that are directly involved in the regulation of the endothelial barrier. We recently performed a comprehensive analysis of the effect of all RhoGTPases and their regulators on basal endothelial integrity. In addition to novel positive regulators of endothelial barrier function, we also identified novel negative regulators, of which the ArhGAP45 (also known as HMHA1) was the most significant. We now demonstrate that ArhGAP45 acts as a Rac-GAP (GTPase-Activating Protein) in endothelial cells, which explains its negative effect on endothelial barrier function. Silencing ArhGAP45 not only promotes basal endothelial barrier function, but also increases cellular surface area and induces sprout formation in a 3D-fibrin matrix. Our data further shows that loss of ArhGAP45 promotes migration and shear stress adaptation. In conclusion, we identify ArhGAP45 (HMHA1) as a novel regulator, which contributes to the fine-tuning of the regulation of basal endothelial integrity.

A CDC42-centered signaling unit is a dominant positive regulator of endothelial integrity.

Endothelial barrier function is carefully controlled to protect tissues from edema and damage inflicted by extravasated leukocytes. RhoGTPases, in conjunction with myriad regulatory proteins, exert both positive and negative effects on the endothelial barrier integrity. Precise knowledge about the relevant mechanisms is currently fragmented and we therefore performed a comprehensive analysis of endothelial barrier regulation by RhoGTPases and their regulators. Combining RNAi with electrical impedance measurements we quantified the relevance of 270 Rho-associated genes for endothelial barrier function. Statistical analysis identified 10 targets of which six promoted- and four reduced endothelial barrier function upon downregulation. We analyzed in more detail two of these which were not previously identified as regulators of endothelial integrity. We found that the Rac1-GEF (Guanine nucleotide Exchange Factor) TIAM2 is a positive regulator and the Cdc42(Rac1)-GAP (GTPase-Activating Protein) SYDE1 is a negative regulator of the endothelial barrier function. Finally, we found that the GAP SYDE1 is part of a Cdc42-centered signaling unit, also comprising the Cdc42-GEF FARP1 and the Cdc42 effector PAK7 which controls the integrity of the endothelial barrier. In conclusion, using a siRNA-based screen, we identified new regulators of barrier function and found that Cdc42 is a dominant positive regulator of endothelial integrity.

Sequence characterization of a sheep cDNA for antithrombin III.

We determined the cDNA sequence of the mRNA for antithrombin III (AT III) from sheep liver. It encodes a protein of 465 amino acids, including a signal peptide of 32 amino acids. The amino acid sequence of the mature protein shows a sequence identity of 89.1%, 95.6% and 85.0% to the human, bovine and rabbit equivalents, respectively. Cysteine residues involved in disulfide bonds as well as potential glycosylation sites are conserved between the four species. In contrast, the amino acid sequence of the signal peptide shows a smaller identity, i.e., 68.7% and 56.3% compared to the human and rabbit preprotein, respectively.

Expression and localization of NOX2 and NOX4 in primary human endothelial cells.

Reactive oxygen species (ROS) control the integrity of the vascular endothelium. Our laboratory has recently shown that transduction of human umbilical vein endothelial cells (HUVECs) with an active variant of the small GTPase Rac promotes the production of ROS, ROS-dependent activation of p38 mitogen-activated protein kinase, and loss of vascular/endothelial-cadherin-mediated cell-cell adhesion. Here we show that HUVECs express mRNAs for NOX2 as well as NOX4 mRNA, but not for NOX1 or NOX3. Interestingly, NOX4 was expressed at 100-fold higher levels compared with NOX2. NOX4-green fluorescent protein largely localizes to an intracellular compartment that costained with a marker for the endoplasmic reticulum, and its distribution did not overlap with lysosomes, Weibel-Palade bodies, or mitochondria. The NOX2-regulatory proteins p47(phox) and p67(phox) associated with the actin cytoskeleton and were found in cell protrusions and membrane ruffles, colocalizing with Rac1. This translocation to the cell periphery was promoted by tumor necrosis factor (TNF)-alpha. Finally, scavenging of ROS was found to impair TNF-alpha-induced cytoskeletal rearrangements and the formation of a confluent endothelial monolayer. Together, these data prove the differential mRNA expression of NOX family members in human endothelium and indicate that these NOX proteins and their regulators may be involved in the control of endothelial cell spreading, motility, and cell-cell adhesion.

Platelet-monocyte complexes support monocyte adhesion to endothelium by enhancing secondary tethering and cluster formation.

OBJECTIVE: Adhesion of monocytes to endothelium can be supported by monocyte-monocyte interactions resulting in the formation of cell aggregates at the vessel wall (clusters). Since platelets that are bound to the injured vessel wall support monocyte adhesion and platelet activation in the circulation leads to formation of platelet-monocyte complexes (PMCs), we examined whether adhesion of PMCs to the vessel wall enhances monocyte clustering. METHODS AND RESULTS: The effect of PMC formation in monocyte adhesion and clustering on human umbilical vein endothelial cells (HUVECs) was studied in vitro with a perfusion system. In the presence of 10% to 20% PMCs, monocyte adhesion and cluster formation to stimulated HUVECs increased 2-fold above levels obtained with pure monocytes. While the observed effects increased with higher PMC levels, blocking-monoclonal antibodies directed against platelet-associated P-selectin or monocyte P-selectin glycoprotein ligand-1 (PSGL-1) reversed adhesion and clustering to control values. In the presence of PMCs, blocking L-selectin decreased adhesion by 25%. When PMCs were present, clustering was only supported by L-selectin at higher shear. These data indicate that monocyte adhesion to the vessel wall is enhanced by PMC-mediated monocyte secondary tethering. These interactions are mainly mediated by P-selectin and PSGL-1. CONCLUSIONS: PMCs in the circulation might be proatherogenic, and prevention of their formation is a possible therapeutic goal.

Small GTP-binding protein Ral modulates regulated exocytosis of von Willebrand factor by endothelial cells.

Weibel-Palade bodies are endothelial cell-specific organelles, which contain von Willebrand factor (vWF), P-selectin, and several other proteins. Recently, we found that the small GTP-binding protein Ral is present in a subcellular fraction containing Weibel-Palade bodies. In the present study, we investigated whether Ral is involved in the regulated exocytosis of Weibel-Palade bodies. Activation of endothelial cells by thrombin resulted in transient cycling of Ral from its inactive GDP-bound to its active GTP-bound state, which coincided with release of vWF. Ral activation and exocytosis of Weibel-Palade bodies were inhibited by incubation with trifluoperazine, an inhibitor of calmodulin, before thrombin stimulation. Functional involvement of Ral in exocytosis was further investigated by the expression of constitutively active and dominant-negative Ral variants in primary endothelial cells. Introduction of active Ral G23V resulted in the disappearance of Weibel-Palade bodies from endothelial cells. In contrast, the expression of the dominant-negative Ral S28N did not affect the amount of Weibel-Palade bodies in transfected cells. These results indicate that Ral is involved in regulated exocytosis of Weibel-Palade bodies by endothelial cells.

Sequential migration of neutrophils across monolayers of endothelial and epithelial cells.

In the course of granulocyte-dominated lung inflammation, granulocytes migrate across the endothelium and epithelium of the lung and cause severe tissue damage. To study this process in more detail, we developed a bilayer transmigration model composed of primary human endothelial and lung epithelial cells, simultaneously cultured on opposite sides of Transwell filters. Electron microscopical analysis showed that the morphology of the cells and the expression of junctional proteins remained unaltered and that matrix components were deposited onto the filter. Intriguingly, neutrophil migration was more efficient across the bilayers than across single epithelial monolayers and did not differ from migration across single endothelial monolayers. Coculture experiments showed that endothelial cells stimulated epithelial cells to release IL-6 and that epithelial cells enhanced release of IL-8 from endothelial cells. Together these data reveal bidirectional signaling and enhanced neutrophil migration in a transmigration model of primary human epithelial and endothelial cells.

Inhibin in immature rat Sertoli cell conditioned medium: a 32 kDa alpha beta-B dimer.

Conditioned medium of cultured Sertoli cells from 21-day-old rats was used as starting material for the isolation of inhibin. Inhibin activity was monitored by the dose dependent suppression of the follicle-stimulating hormone release of cultured rat pituitary cells. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis analysis of the highly purified inhibin preparation revealed a 32 kDa protein after silver staining, which could be separated in subunits of 18 kDa and 12 kDa after reduction. Western blot analysis with an antibody recognizing the 22 N-terminal amino acids of the alpha-subunit of 32 kDa bovine inhibin confirmed the presence of a 32 kDa inhibin molecule under non-reducing conditions, whereas an 18 kDa alpha-subunit was found after reduction. An antibody recognizing the beta-A subunit of inhibin did not yield a signal after Western blotting. N-terminal amino acid sequence analysis of two highly purified preparations of inhibin obtained using different methods yielded the sequence predicted for a 32 kDa alpha beta-B dimer on basis of cDNA nucleotide sequence. This result is in agreement with the large excess of beta-B over beta-A mRNA in the rat testis.

The neuropeptide schistosomin and haemolymph from parasitized snails induce similar changes in excitability in neuroendocrine cells controlling reproduction and growth in a freshwater snail.

Infection of the snail Lymnaea stagnalis with the schistosome parasite Trichobilharzia ocellata results in inhibition of reproduction and in giant growth. Parasite-related effects on the neuroendocrine centres that control these processes were studied electrophysiologically. Haemolymph from infected snails reduced the excitability of the caudodorsal cells, which control egg laying. In contrast, the excitability of the growth-controlling Light Green Cells was increased under these conditions. The endogenous anti-gonadotropic neuropeptide schistosomin, the presence of which is strongly enhanced in parasitized snails, induced similar effects. Schistosomin apparently plays an important role in the balance between reproduction and growth in Lymnaea. This balance is severely disturbed during parasitic infection, probably as a result of the release of the peptide.

Gi-mediated activation of the Ras/MAP kinase pathway involves a 100 kDa tyrosine-phosphorylated Grb2 SH3 binding protein, but not Src nor Shc.

Mitogenic G protein-coupled receptors, such as those for lysophosphatidic acid (LPA) and thrombin, activate the Ras/MAP kinase pathway via pertussis toxin (PTX)-sensitive Gi, tyrosine kinase activity and recruitment of Grb2, which targets guanine nucleotide exchange activity to Ras. Little is known about the tyrosine phosphorylations involved, although Src activation and Shc phosphorylation are thought to be critical. We find that agonist-induced Src activation in Rat-1 cells is not mediated by Gi and shows no correlation with Ras/MAP kinase activation. Furthermore, LPA-induced tyrosine phosphorylation of Shc is PTX-insensitive and Ca2+-dependent in COS cells, but undetectable in Rat-1 cells. Expression of dominant-negative Src or Shc does not affect MAP kinase activation by LPA. Thus, Gi-mediated Ras/MAP kinase activation in fibroblasts and COS cells involves neither Src nor Shc. Instead, we detect a 100 kDa tyrosine-phosphorylated protein (p100) that binds to the C-terminal SH3 domain of Grb2 in a strictly Gi- and agonist-dependent manner. Tyrosine kinase inhibitors and wortmannin, a phosphatidylinositol (PI) 3-kinase inhibitor, prevent p100-Grb2 complex formation and MAP kinase activation by LPA. Our results suggest that the p100-Grb2 complex, together with an upstream non-Src tyrosine kinase and PI 3-kinase, couples Gi to Ras/MAP kinase activation, while Src and Shc act in a different pathway.

The Light Green Cells of Lymnaea: a neuroendocrine model system for stimulus-induced expression of multiple peptide genes in a single cell type.

We review recent experiments showing that the cerebral neuroendocrine Light Green Cells (LGCs) of the freshwater snail, Lymnaea stagnalis, express a family of distinct though related molluscan insulin-related peptide (MIP) genes. The LGCs are involved in the regulation of a wide range of interrelated life processes associated with growth, (energy) metabolism and reproduction. We consider the mechanism of generation of diversity among MIPs, and present evidence that conditions with distinct effects on growth, metabolism and reproduction also can induce distinct patterns of expression of the MIP and schistosomin genes. The stimulus-dependent expression of multiple neuropeptide genes enormously increases the adaptive potential of a peptidergic neuron. We suggest that this contributes significantly to the information-handling capacity of the brain.

Gi-mediated activation of the p21ras-mitogen-activated protein kinase pathway by alpha 2-adrenergic receptors expressed in fibroblasts.

The alpha 2-adrenergic receptors are linked to inhibition of adenylylcyclase and, under certain circumstances, to stimulation of phospholipid hydrolysis via pertussis toxin-sensitive G proteins. Here we show that alpha 2-adrenergic receptors can couple to an alternative signaling pathway. When expressed in Rat-1 cells, stimulation of the alpha 2A receptor, which couples to Gi2 and Gi3, causes rapid, transient activation of the protooncogene product p21ras as measured by an increase in the amount of bound GTP. Furthermore, alpha 2A receptor stimulation causes rapid phosphorylation of the p42 mitogen-activated protein (MAP) kinase. Pertussis toxin completely inhibits both p21ras activation and MAP kinase phosphorylation, but both responses appear to be independent of adenylylcyclase inhibition or phospholipase stimulation. Thus, alpha 2-adrenergic receptors can couple to the p21ras-MAP kinase pathway via Gi, which may explain the mitogenic potential of alpha 2 agonists in certain cell types; together with previous results, these findings further suggest that activation of this pivotal signaling pathway may be a common event in the action of Gi-coupled receptors.

Involvement of Fcgamma receptors and beta2 integrins in neutrophil activation by anti-proteinase-3 or anti-myeloperoxidase antibodies.

We previously described the requirement of tumour necrosis factor-alpha (TNF-alpha) and the role of beta2 integrins in the Fc-gamma receptor IIa (FcgammaRIIa)-mediated mechanism of neutrophil activation by antiproteinase-3 (anti-PR3) or anti-myeloperoxidase (anti-MPO) antibodies. In the present study, we assessed the involvement of FcgammaRIIIb by studying the respiratory burst activation of completely FcgammaRIIIb-deficient neutrophils primed by TNF-alpha and exposed to anti-PR3 or anti-MPO. Activation of the NADPH oxidase occurred normally in these neutrophils, which indicates that engagement of FcgammaRIIIb is not essential in our model. Experiments performed with neutrophils from severe leucocyte adhesion deficiency (LAD) patients confirmed that beta2 integrins play a pivotal role in this activation. We next studied whether adhesion per se, beta2-integrin-mediated adhesion, or beta2-integrin ligation without adhesion is necessary or sufficient for this activation. Anti-PR3 or anti-MPO induced an FcgammaRIIa-dependent burst in TNF-primed neutrophils incubated in wells coated with poly-L-lysine, known to induce beta2-integrin-independent adhesion, but this reaction was still inhibited by blocking CD18 antibodies. In a system with granulocyte-macrophage colony-stimulating factor (GM-CSF)-primed neutrophils, which did not enhance adhesion, we measured a similar activation by anti-PR3 or anti-MPO and inhibition by CD18. We also noticed that treatment with the beta2-integrin-activating CD18 MoAb KIM185 per se is insufficient for neutrophil activation by anti-PR3 or anti-MPO. We therefore conclude that ligation of beta2 integrins rather than adherence per se is essential for this activation, and that TNF-alpha or GM-CSF is needed for priming but not for adherence.