Rottlerin causes pulmonary edema in vivo: a possible role for PKCdelta.

In the present study, we assessed the effects of chemical inhibitors shown to be selective for protein kinase C (PKC) isoforms on lung barrier function both in vitro and in vivo. Rottlerin, a purported inhibitor of PKCdelta, but not other chemical inhibitors, dose dependently promoted barrier dysfunction in lung endothelial cells in vitro. This barrier dysfunction correlated with structural changes in focal adhesions and stress fibers, which were consistent with functional changes in cell stiffness. To determine whether the effects noted in vitro correlated with changes in intact lungs, we tested the effects of rottlerin in the formation of pulmonary edema in rats using both ex vivo and in vivo models. Isolated, perfused lungs demonstrated a significant increase in filtration coefficients on exposure to rottlerin, compared with vehicle-treated lungs, an effect that correlated with increased extravasation of Evan's blue dye (EBD)-conjugated albumin. Additionally, compared with vehicle, the ratio of the wet lung weights to dry lung weights was significantly greater on exposure of animals to rottlerin; rottlerin also produced a dose-dependent increase in EBD extravasation into the lungs. These effects on lung edema occurred without any increase in right ventricular pressures. Microscopic assessment of edema in the ex vivo lungs demonstrated perivascular cuffing, with no evidence of septal capillary leak, in rottlerin-exposed lungs. Taken together, rottlerin increases barrier dysfunction in pulmonary endothelial cell monolayers and causes pulmonary edema in rats; results suggestive of an important role for PKCdelta in maintaining lung endothelial barrier function.     

The role of arachidonic acid in vasogenic brain edema

Arachidonic acid is released rapidly from cellular membrane phospholipids after pathological insults associated with the delayed development of brain edema. Intracerebral injection of arachidonic acid caused significant increases in brain water and sodium content with decreases in potassium content and Na+,K+-ATPase activity. The 125I-labeled bovine serum albumin spaces in brain (a measure of blood-brain barrier permeability) rose threefold 24 h after arachidonic acid injection. There was gross and microscopic evidence of edema. Saturated fatty acids and monounsaturated fatty acids were not effective. These data indicate that the endothelial cells of the blood-brain barrier are target sites for the action of arachidonic acid. It is hypothesized that the increased permeability of endothelial cells to macromolecules and water results from alterations of membrane phospholipids and increased vesicular transport, changes that are responsible for the delayed development of vasogenic edema.     

Hydrocortisone decreases retinal endothelial cell water and solute flux coincident with increased content and decreased phosphorylation of occludin

Corticosteroids provide an effective treatment to reduce edema for conditions in which the blood-brain or blood-retinal barrier is compromised. However, little is known about the mechanism by which these hormones affect endothelial cell function. We hypothesized that hydrocortisone would reduce transport of water and solutes across bovine retinal endothelial cell (BREC) monolayers coincident with changes to the tight junction protein occludin. Treatment of BREC with 103 nm hydrocortisone for two days significantly decreased water and solute transport across cell monolayers. Immunoblot analysis of occludin extracted in SDS or urea based buffers revealed a 1.65- or 2.57-fold increase in content, respectively. A similar two-fold increase in occludin mRNA was observed by real-time PCR. Immunocytochemistry revealed hydrocortisone dramatically increased both occludin and ZO-1 staining at the cell border. Additionally, 4 h of hydrocortisone treatment significantly reduced occludin phosphorylation. To our knowledge, this is the first example of a regulated decrease in occludin phosphorylation associated with increased barrier properties. In conclusion, hydrocortisone directly affects retinal endothelial cell barrier properties coincident with changes in occludin content, phosphorylation and tight junction assembly. Localized hydrocortisone therapy may be developed as a treatment option for patients suffering from retinal edema due to diabetes.     

Cytotoxic effects of 3-methylindole on alveolar epithelial cells and macrophages: with special reference to microtubular and filamentous assemblies in alveolar type I cells of bovine lung

The alveolar type I cell is a major permeability barrier between the pulmonary interstitium and alveolar spaces and its thin cytoplasmic processes are greatly susceptible to injury. These cells are often observed to undergo progressive vesiculation, vacuolization and desquamation during 3-methylindole (3MI)-induced acute pulmonary edema after oral administration in goats and cattle. The present study describes proliferation of SER and the presence of polymerized tubulin in the form of microtubules arranged in large bundles shown at ultrastructural level as well as with immunofluorescence staining for tubulin in alveolar type I cells 72 hours after 3MI treatment. Such changes were not seen in pulmonary endothelial cells, alveolar type II cells, alveolar macrophages and neutrophils. The possible role of microtubules in alveolar type I cells as a mechanistic support to resist disruption against the forces of interstitial and alveolar edema is compared with alveolar type II cells, alveolar macrophages and neutrophils. The latter cells undergo dynamic movements in response to inflammatory stimuli and therefore did not show microtubules in their cytoplasm.     

Post-translational modifications of S1PR1 and endothelial barrier regulation

Sphingosine-1-phosphate receptor-1 (S1PR1), a G-protein coupled receptor that is expressed in endothelium and activated upon ligation by the bioactive lipid sphingosine-1-phosphate (S1P), is an important vascular-barrier protective mechanism at the level of adherens junctions (AJ). Loss of endothelial barrier function is a central factor in the pathogenesis of various inflammatory conditions characterized by protein-rich lung edema formation, such as acute respiratory distress syndrome (ARDS). While several S1PR1 agonists are available, the challenge of arresting the progression of protein-rich edema formation remains to be met. In this review, we discuss the role of S1PRs, especially S1PR1, in regulating endothelial barrier function. We review recent findings showing that replenishment of the pool of cell-surface S1PR1 may be crucial to the effectiveness of S1P in repairing the endothelial barrier. In this context, we discuss the S1P generating machinery and mechanisms that regulate S1PR1 at the cell surface and their impact on endothelial barrier function.     

Endothelial cells as early sensors of pulmonary interstitial edema.

We studied responses of endothelial and epithelial cells in the thin portion of the air-blood barrier to a rise in interstitial pressure caused by an increase in extravascular water (interstitial edema) obtained in anesthetized rabbits receiving saline infusion (0.5 ml.kg(-1).min(-1) for 3 h). We obtained morphometric analyses of the cells and of their microenvironment (electron microscopy); furthermore, we also studied in lung tissue extracts the biochemical alterations of proteins responsible for signal transduction (PKC, caveolin-1) and cell-cell adhesion (CD31) and of proteins involved in membrane-to-cytoskeleton linkage (alpha-tubulin and beta-tubulin). In endothelial cells, we observed a folding of the plasma membrane with an increase in cell surface area, a doubling of plasmalemma vesicular density, and an increase in cell volume. Minor morphological changes were observed in epithelial cells. Edema did not affect the total plasmalemma amount of PKC, beta-tubulin, and caveolin-1, but alpha-tubulin and CD-31 increased. In edema, the distribution of these proteins changed between the detergent-resistant fraction of the plasma membrane (DRF, lipid microdomains) and the rest of the plasma membrane [high-density fractions (HDFs)]. PKC and tubulin isoforms shifted from the DRF to HDFs in edema, whereas caveolin-1 increased in DRF at the expense of a decrease in phosphorylated caveolin-1. The changes in cellular morphology and in plasma membrane composition suggest an early endothelial response to mechanical stimuli arising at the interstitial level subsequently to a modest (approximately 5%) increase in extravascular water.     

Complement proteins C5b-9 induce vesiculation of the endothelial plasma membrane and expose catalytic surface for assembly of the prothrombinase enzyme complex

Assembly of the terminal complement proteins C5b-9 on human endothelial cells results in increased cytosolic calcium and nonlytic secretion of high molecular weight multimers of von Willebrand factor from intracellular storage granules. We now demonstrate that this C5b-9-induced secretory response is accompanied by vesiculation of membrane particles from the endothelial surface which express binding sites for factor Va and support prothrombinase activity. Exposure of factor Va binding sites after C5b-9 assembly was accompanied by greater than 2-fold increase in prothrombinase activity, which was not observed for cells exposed to C5b-8 (in the absence of C9). By contrast, only a 3-16% increase in prothrombinase activity was observed when these cells were maximally stimulated to secrete by either histamine, thrombin, or the Ca2+ ionophore A23187. Increased prothrombinase activity after C5b-9 was not accompanied by a change in thrombomodulin activity, and was unrelated to cell lysis, the complement-treated cells remaining greater than 99% viable. Endothelial prothrombinase activity was predominately associated with small membrane vesicles (less than 1 microns diameter) released from the cell monolayer. Analysis by fluorescence-gated flow cytometry revealed that these vesicles incorporate the C5b-9 proteins and express binding sites for factor Va. The capacity of the C5b-9 proteins to induce vesiculation of the endothelial plasma membrane and thereby expose catalytic surface for the prothrombinase enzyme complex may contribute to fibrin deposition associated with immune endothelial injury.     

Endothelial domes encapsulate adherent neutrophils and minimize increases in vascular permeability in paracellular and transcellular emigration

Local edema, a cardinal sign of inflammation associates closely with neutrophil emigration. Neutrophil emigration has been described to occur primarily through endothelial junctions (paracellular) and more rarely directly through endothelial cells (transcellular). Recently, we reported that unlike in wild-type (wt) mice, Mac-1-/- (CD11b) neutrophils predominantly emigrated transcellularly and was significantly delayed taking 20-30 min longer than the paracellular emigration (wt). In the present study we noted significant anatomical disruption of the endothelium and hypothesized that transcellular emigration would greatly increase vascular permeability. Surprisingly, despite profound disruption of the endothelial barrier as the neutrophils moved through the cells, the changes in vascular permeability during transcellular emigration (Mac-1-/-) were not increased more than in wt mice. Instead increased vascular permeability completely tracked the number of emigrated cells and as such, permeability changes were delayed in Mac-1-/- mice. However, by 60 min neutrophils from both sets of mice were emigrating in large numbers. Electron-microscopy and spinning disk multichannel fluorescence confocal microscopy revealed endothelial docking structures that progressed to dome-like structures completely covering wt and Mac-1-/- neutrophils. These domes completely enveloped the emigrating neutrophils in both wt and Mac-1-/- mice making the mode of emigration underneath these structures extraneous to barrier function. In conclusion, predominantly paracellular versus predominantly transcellular emigration does not affect vascular barrier integrity as endothelial dome-like structures retain barrier function.     

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.     

The severity of shock is associated with impaired rates of net alveolar fluid clearance in clinical acute lung injury

The rate of alveolar fluid clearance (AFC) is associated with mortality in clinical acute lung injury (ALI). Patients with ALI often develop circulatory shock, but how shock affects the rate of AFC is unknown. To determine the effect of circulatory shock on the rate of AFC in patients with ALI, the rate of net AFC was measured in 116 patients with ALI by serial sampling of pulmonary edema fluid. The primary outcome was the rate of AFC in patients with shock compared with those without shock. We also tested the effects of shock severity and bacteremia. Patients with ALI and shock (n = 86) had significantly slower rates of net AFC compared with those without shock (n = 30, P = 0.03), and AFC decreased significantly as the number of vasopressors increased. Patients with positive blood cultures (n = 21) had slower AFC compared with patients with negative blood cultures (n = 96, P = 0.023). In addition, the edema fluid-to-plasma protein ratio, an index of alveolar-capillary barrier permeability, was highest in patients requiring the most vasopressors (P < 0.05). Patients with ALI complicated by circulatory shock and bacteremia had slower rates of AFC compared with patients without shock or bacteremia. An impaired capacity to reabsorb alveolar edema fluid may contribute to high mortality among patients with sepsis-induced ALI. These findings also suggest that vasopressor use may be a marker of alveolar-capillary barrier permeability in ALI and provide justification for new therapies that enhance alveolar epithelial and endothelial barrier integrity in ALI, particularly in patients with shock.     

Effects of hypoxia-reoxygenation on rat blood-brain barrier permeability and tight junctional protein expression

Cerebral microvessel endothelial cells that form the blood-brain barrier (BBB) have tight junctions (TJs) that are critical for maintaining brain homeostasis. The effects of initial reoxygenation after a hypoxic insult (H/R) on functional and molecular properties of the BBB and TJs remain unclear. In situ brain perfusion and Western blot analyses were performed to assess in vivo BBB integrity on reoxygenation after a hypoxic insult of 6% O2 for 1 h. Model conditions [blood pressure, blood gas chemistries, cerebral blood flow (CBF), and brain ATP concentration] were also assessed to ensure consistent levels and criteria for insult. In situ brain perfusion revealed that initial reoxygenation (10 min) significantly increased the uptake of [14C]sucrose into brain parenchyma. Capillary depletion and CBF analyses indicated the perturbations were due to increased paracellular permeability rather than vascular volume changes. Hypoxia with reoxygenation (10 min) produced an increase in BBB permeability with associated alterations in tight junctional protein expression. These results suggest that H/R leads to reorganization of TJs and increased paracellular diffusion at the BBB, which is not a result of increased CBF, vascular volume change, or endothelial uptake of marker. Additionally, the tight junctional protein occludin had a shift in bands that correlated with functional changes (i.e., increased permeability) without significant change in expression of claudin-3, zonula occludens-1, or actin. H/R-induced changes in the BBB may result in edema and/or associated pathological outcomes.     

Antigen presentation by endothelial cells: what role in the pathophysiology of malaria?

Disruption of the endothelial cell (EC) barrier leads to pathology via edema and inflammation. During infections, pathogens are known to invade the EC barrier and modulate vascular permeability. However, ECs are semi-professional antigen-presenting cells, triggering T-cell costimulation and specific immune-cell activation. This in turn leads to the release of inflammatory mediators and the destruction of infected cells by effectors such as CD8(+) T-cells. During malaria, transfer of parasite antigens to the EC surface is now established. At the same time, CD8 activation seems to play a major role in cerebral malaria. We summarize here some of the pathways leading to antigen presentation by ECs and address the involvement of these mechanisms in the pathophysiology of cerebral malaria.     

Intracerebral VEGF injection highly upregulates AQP4 mRNA and protein in the perivascular space and glia limitans externa

Stroke is the third leading cause of death and the leading cause of adult disability in the industrialized nations. One of the consequences of stroke is blood-brain barrier (BBB) leakage and subsequent edema, which is one of the causes of mortality in this pathology. Aquaporin-4 (AQP4) is the most abundant water channel in the brain. Studies in AQP4 knock-out mice have shown a prominent role of this water channel in edema development and resolution after ischemia. Here we have studied changes in AQP4 mRNA and protein expression in response to vascular endothelial growth factor (VEGF), a potent angiogenic factor. VEGF administration highly upregulated AQP4 mRNA and protein in the ventral midbrain. Perfusion of the animals with FITC-albumin prior to sacrifice demonstrated localization of AQP4 protein in close proximity to the VEGF-induced new blood vessels. Expression levels of AQP4 mRNA were maximum 7 days after VEGF injection whereas our previous report showed that BBB leakage is resolved at this time point. Therefore, we speculate a positive role of AQP4 in edema resolution, which may partially explain the previously reported beneficial effects of delayed VEGF administration in ischemic rats. Our results provide new insights into the molecular changes in the edematous brain and may help in future therapeutical directions.     

Anthrax lethal toxin disrupts the endothelial permeability barrier through blocking p38 signaling

Exposure to anthrax causes life-threatening disease through the action of the toxin produced by the Bacillus anthracis bacteria. Lethal factor (LF), an anthrax toxin component which causes severe vascular leak and edema, is a protease which specifically degrades MAP kinase kinases (MKK). We have recently shown that p38 MAP kinase activation leading to HSP27 phosphorylation augments the endothelial permeability barrier. We now show that treatment of rat pulmonary microvascular endothelial cells with anthrax lethal toxin (LeTx), which is composed of LF and the protective antigen, increases endothelial barrier permeability and gap formation between endothelial cells through disrupting p38 signaling. LeTx treatment increases MKK3b degradation and in turn decreases p38 activity at baseline as well as after activation of p38 signaling. Consequently, LeTx treatment decreases activation of the p38 substrate kinase, MK2, and the phosphorylation of the latter's substrate, HSP27. LeTx treatment disrupts other signaling pathways leading to suppression of Erk-mediated signaling, but these effects do not correlate with LeTx-induced barrier compromise. Overexpressing phosphomimicking (pm)HSP27, which protects the endothelial permeability barrier against LeTx, blocks LeTx inactivation of p38 and MK2, but it does not block MKK3b degradation or Erk inactivation. Our results suggest that LeTx might cause vascular leak through inactivating p38-MK2-HSP27 signaling and that activating HSP27 phosphorylation specifically restores p38 signaling and blocks anthrax LeTx toxicity. The fact that barrier integrity could be restored by pmHSP27 overexpression without affecting degradation of MKK3b, or inactivation of Erk, suggests a specific and central role for p38-MK2-HSP27 in endothelial barrier permeability regulation.     

Compositional changes in lipid microdomains of air-blood barrier plasma membranes in pulmonary interstitial edema.

We evaluated in anesthetized rabbits the compositional changes of plasmalemmal lipid microdomains from lung tissue samples after inducing pulmonary interstitial edema (0.5 ml/kg for 3 h, leading to approximately 5% increase in extravascular water). Lipid microdomains (lipid rafts and caveolae) were present in the detergent-resistant fraction (DRF) obtained after discontinuous sucrose density gradient. DRF was enriched in caveolin-1, flotillin, aquaporin-1, GM1, cholesterol, sphingomyelin, and phosphatidylserine, and their contents significantly increased in interstitial edema. The higher DRF content in caveolin, flotillin, and aquaporin-1 and of the ganglioside GM1 suggests an increase both in caveolar domains and in lipid rafts, respectively. Compositional changes could be ascribed to endothelial and epithelial cells that provide most of plasma membrane surface area in the air-blood barrier. Alterations in lipid components in the plasma membrane may reflect rearrangement of floating lipid platforms within the membrane and/or lipid translocation from intracellular stores. Lipid traffic could be stimulated by the marked increase in hydraulic interstitial pressure after initial water accumulation, from approximately -10 to 5 cmH2O, due to the low compliance of the pulmonary tissue, in particular in the basement membranes and in the interfibrillar substance. Compositional changes in lipid microdomains represent a sign of cellular activation and suggest the potential role of mechanotransduction in response to developing interstitial edema.     

Integration of Biochemical and Biomechanical Signals Regulating Endothelial Barrier Function

Endothelial barrier function is critical for tissue homeostasis throughout the body. Disruption of the endothelial monolayer leads to edema, vascular diseases and even cancer metastasis among other pathological conditions. Breakdown of the endothelial barrier integrity triggered by cytokines (e.g.IL-8,IL-1β) and growth factors (e.g.VEGF) is well documented. However, endothelial cells are subject to major biomechanical forces that affect their behavior. Due to their unique location at the interface between circulating blood and surrounding tissues, endothelial cells experience shear stress, strain and contraction forces. More than three decades ago, it was already appreciated that shear flow caused endothelial cells alignment in the direction of the flow. After that observation, it took around 20 years to begin to uncover some of the mechanisms used by the cells for mechanotransduction. In this review, we describe mechanosensors on the endothelium identified to date and the associated signaling pathways that integrate biochemical and biomechanical inputs into biological responses and how they modulate the integrity of the endothelial barrier.     

Correlation of permeability with the structure of the endothelial layer of pulmonary artery intimal explants

Changes in vascular permeability are associated with structural damage to endothelial cells. These functional and structural changes can be produced experimentally and examined by using intimal explants from bovine pulmonary artery. Correlation of functional with structural changes allows us to dissect the mechanisms responsible for endothelial damage. We have shown that incubation of intimal explants with histamine causes transient formation of interendothelial dilatations and an increased rate of equilibration of tritiated water and [14C]sucrose across the intimal explant. Exposure to endotoxin also causes interendothelial dilatations but the endothelial damage is more severe than that with histamine, and in vivo experiments show a more prolonged increase in pulmonary vascular permeability. Leukocyte migration has also been suggested to result in a decreased barrier function of the endothelial layer. Experiments with the endothelial layer of intimal explants and separated bovine leukocytes suggest that transendothelial migration may depend on the chemotactic stimulus. Neither granulocyte migration toward zymosan-activated plasma nor lymphocyte migration toward lymphocyte-conditioned medium (RPMI in which lymphocytes were incubated with concanavalin A) leads to detectable increases in explant permeability, but granulocyte migration toward lymphocyte-conditioned medium does result in increased equilibration of [14C]sucrose. Finally, a theoretical model has been used to examine the permeability changes seen for the intimal explants exposed to histamine. The model consists of two compartments with radioactive tracers diffusing across a filter of known permeability. Such a model gives good agreement with data obtained in intact sheep, indicating that mathematical models allow quantitative estimates of barrier function in intimal explants that compare favorably with in vivo data.     

蛋白激酶在微血管通透性中的作用

微血管内皮细胞层是一层半选择通透性屏障,可以调节血液中的液体、溶质和血浆蛋白进入组织间隙。在炎症刺激作用下,可通过旁细胞途径和跨细胞途径引起内皮通透性上升。旁细胞通路主要由内皮细胞间的紧密连接、黏附连接和细胞与外基质的黏着斑组成。炎症介质,如脂多糖和肿瘤坏死因子α可激活多种蛋白激酶。活化的蛋白激酶主要包括Rho相关的卷曲蛋白激酶、肌球蛋白轻链激酶、蛋白激酶C、酪氨酸激酶和丝裂原活化蛋白激酶等,参与引发内皮屏障生化和结构改变,旁细胞通路开放,导致通透性上升。该文对上述蛋白激酶在微血管通透性中作用机制的研究进展进行综述。     

Alterations of bone marrow sinus endothelium induced by ionizing irradiation: implications in the homing of intravenously transplanted marrow cells.

The endothelium of bone marrow sinuses is a continuous layer which is selective in its cellular transport. It is not known how selective and massive seeding of hemopoietic progenitor cells after intravenous transplantation of marrow cells occurs. We postulate that the conditioning irradiation could disrupt the endothelial barrier, thus permitting the "homing" of progenitor cells to occur. To demonstrate this phenomenon, we irradiated mice with doses ranging from 100-2000 cGy-total body and studied perfusion-fixed marrow by transmission electron microscopy. The major finding was sloughing and denudation of plasma membrane, particularly on the luminal side of endothelium. Membrane vesiculation was also frequently seen in this border. Moreover, dilatation of the perinuclear space and rough endoplasmic reticulum was commonplace and testified to instability and fragility of the membrane system. Focal cytoplasmic swelling of endothelium was seen reflecting increased permissiveness of the endothelial barrier. Endocytosis and phagocytosis were increased in the marrow; and the endothelium, normally quiescent with regard to phagocytosis, was now overtly phagocytic. A dipogenecity of the adventitial layer was increased as hemopoietic function of marrow decreased. The end result of membrane alterations in the endothelium was the appearance of discontinuities in these cells, which form the essential element of bone marrow-blood barrier. Consequent to these discontinuities, the permissiveness of the endothelial barrier was enhanced and those cellular elements, such as mature, nonreticulated erythrocytes that are normally confined to the vascular space, now appeared in large number in the hemopoietic compartment. With low doses, these findings were transient and repair set in by 1-2 weeks. With higher doses, total disruption of marrow-blood barrier occurred and the process did not seem to be repairable. We conclude that the conditioning irradiation before bone marrow transplantation is essential in disrupting the endothelial barrier and permitting large-scale entry of transplanted cells into the hemopoietic compartment.