Adrenomedullin Reduces Endothelial Hyperpermeability
Endothelial hyperpermeability induced by inflammatory mediators is a hallmark of sepsis and adult respiratory distress syndrome. Increased levels of the regulatory peptide adrenomedullin (ADM) have been found in patients with systemic inflammatory response. We analyzed the effect of ADM on the permeability of cultured human umbilical vein endothelial cell (HUVEC) and porcine pulmonary artery endothelial cell monolayers. ADM dose-dependently reduced endothelial hyperpermeability induced by hydrogen peroxide (H2O2), thrombin, and Escherichia coli hemolysin. Moreover, ADM pretreatment blocked H2O2-related edema formation in isolated perfused rabbit lungs and increased cAMP levels in lung perfusate. ADM bound specifically to HUVECs and porcine pulmonary artery endothelial cells and increased cellular cAMP levels. Simultaneous inhibition of cAMP-degrading phosphodiesterase isoenzymes 3 and 4 potentiated ADM-dependent cAMP accumulation and synergistically enhanced ADM-dependent reduction of thrombin-induced hyperpermeability. However, ADM showed no effect on endothelial cGMP content, basal intracellular Ca2+ levels, or the H2O2-stimulated, thrombin-stimulated, or Escherichia coli hemolysin–stimulated Ca2+ increase. ADM diminished thrombin- and H2O2-related myosin light chain phosphorylation as well as stimulus-dependent stress fiber formation and gap formation in HUVECs, suggesting that ADM may stabilize the barrier function by cAMP-dependent relaxation of the microfilament system. These findings identify a new function of ADM and point to ADM as a potential interventional agent for the reduction of vascular leakage in sepsis and adult respiratory distress syndrome.
The incidence of sepsis and ensuing multiple organ failure has increased over the past two decades and has caused multiple deaths in intensive care units. The development of adult respiratory distress syndrome (ARDS) characterized by noncardiogenic pulmonary edema contributes substantially to a fatal outcome.1 Increased microvascular permeability is a hallmark of an inflammatory reaction, including ARDS-related pulmonary edema formation. Circumstantial evidence has suggested that endothelial hyperpermeability is related to alterations of the cellular cytoskeleton.2 Endothelial cells have been shown to contain an elaborate microfilament system allowing active actin- and myosin-based cell contraction. Activation of cell contraction and disturbance of junctional organization subsequently result in the induction of interendothelial gaps followed by enhanced paracellular endothelial permeability.2–6⇓⇓⇓⇓ Major initiators of this process are polymorphonuclear leukocyte–derived oxygen metabolites,7–9⇓⇓ pore-forming bacterial exotoxins,9,10⇓ and endogenous proinflammatory mediators, such as thrombin.5,9,11⇓⇓
Exposure of endothelial cells to hydrogen peroxide (H2O2) results in the activation of multiple signaling pathways, finally leading to the activation of myosin light chain (MLC) kinase and disrupted barrier function.7,8⇓ Thrombin-activated Rho kinase increases the phosphorylation of endothelial cell MLC by directly phosphorylating MLC and simultaneously inhibiting MLC phosphatase, thereby maximally enhancing MLC phosphorylation.4,5⇓ The hemolysin of Escherichia coli (HlyA) is an important prototype of pore-forming bacterial exotoxins that allows Ca2+ influx into the cytosol of target cells.9,12,13⇓⇓ All three stimuli (H2O2, thrombin, and HlyA) have been shown to induce endothelial cell contraction followed by endothelial hyperpermeability in vitro and in vivo.
Previous studies from others and our group have demonstrated an improvement of endothelial barrier function by the activation of adenylyl cyclase and/or phosphodiesterase (PDE) inhibition.2,8,9,14⇓⇓⇓ Analysis of the endothelial cell PDE isoenzyme pattern showed high activities of PDE2 to PDE4. PDE2 mainly metabolizes cGMP, whereas PDE3/4 is specific for cAMP.8,9⇓
Accumulating evidence suggests a pivotal role of adrenomedullin (ADM), a multifunctional regulatory peptide, in sepsis and septic shock.15–19⇓⇓⇓⇓ ADM, a 52-amino-acid peptide belonging to the calcitonin gene–related peptide family, participates in the control of central body functions, such as vascular tone regulation or fluid and electrolyte homeostasis (see reviews20,21⇓). In systemic inflammatory response, plasma levels of ADM in vertebrates, including human beings, were found to be elevated.15,17–19⇓⇓⇓ Endothelial cells produce ADM in response to proinflammatory cytokines or lipopolysaccaride22 under the control of the transcription factors nuclear factor for interleukin-6 and activator protein 2.22
Calcitonin receptor–like receptor (CRLR) and receptor activity–modifying protein (RAMP)2 and RAMP3 together form ADM receptors coupled to cholera toxin–sensitive G proteins; however, alternative ADM binding receptors may exist.20,23⇓ Stimulation of virtually all cells currently investigated with ADM has resulted in a marked increase in cellular cAMP content and subsequent activation of protein kinase A (PKA).20,21,24⇓⇓ In contrast, conflicting results regarding an ADM-induced increase in [Ca2+]i have been reported.24,25⇓
Because transgenic mice overexpressing ADM in their vasculature have turned out to be resistant to lipopolysaccharide-induced shock, the role of ADM in sepsis deserves special consideration.26 Moreover, mice with disrupted ADM genes have displayed an extreme hydrops fetalis and cardiovascular abnormalities, suggesting a central role of ADM in the regulation of the cardiovascular system, especially endothelial cell function.27
In the present investigation, we tested the hypothesis that elevated ADM levels in systemic inflammatory response stabilize endothelial barrier function by preventing endothelial cell contraction and paracellular fluid flux. ADM treatment diminished endothelial hyperpermeability induced by stimuli as diverse as oxygen metabolites, pore-forming bacterial exotoxins, and endogenous proinflammatory mediators. Moreover, ADM blocked H2O2-related lung edema formation in a model of isolated rabbit lungs. ADM induced cAMP formation, reduced thrombin- and H2O2-induced MLC phosphorylation, and prevented endothelial cell contraction. Overall, our data suggest that ADM may act as a counterregulatory peptide in systemic inflammatory response by improvement of endothelial barrier function.
Materials and Methods
Preparation of Endothelial Cells
Radioligand Binding for ADM
Competitive receptor binding studies for human ADM were performed with minor modifications as described earlier for neuropeptide Y.30
Determination of HUVEC Cyclic Nucleotide Content
Cyclic nucleotide content was measured by using a commercially available ELISA (Biotrend).
Analysis of Endothelial Permeability
Hydraulic conductivity of endothelial cell monolayers was determined as described previously.8,9,31⇓⇓ Briefly, a confluent cell monolayer on a filter membrane was mounted in a modified chemotaxis chamber, and a hydrostatic pressure of 102 mm H2O was applied to the “luminal” side of the cell monolayer. The filtration rate across the endothelial monolayer was continuously determined, and the hydraulic conductivity was calculated and expressed as 10−5 cm · s−1 · cm H2O−1.
Cells were fixed in paraformaldehyde and permeabilized, and actin was stained by using phalloidin Alexa 488 (Molecular Probes). Cells were analyzed with the use of a Pascal 5 confocal scanning laser microscope (Zeiss).
Detection of MLC
Cells were harvested in lysis buffer containing phosphatase and protease inhibitors. Equal amounts of lysates were subjected to SDS-PAGE (12.5% gel) and blotted. Membranes were simultaneously exposed to goat phospho-specific MLC (Thr18/Ser19) and rabbit extracellular signal–regulated kinase (ERK)1-specific antibody (both Santa Cruz Biotechnology) incubated with secondary antibodies (IRDye 800–labeled anti-goat and Cy5.5-labeled anti-rabbit, respectively). Proteins were detected by using an Odyssey infrared imaging system (LI-COR Inc).
Determination of [Ca2+]i
HUVECs cultured on glass coverslips were loaded with the fluorescent Ca2+-sensitive dye fura 2 and analyzed by use of a fluorescence spectrophotometer (Instruments S.A.). Excitation wavelength was alternated between 343 and 380 nm. Emitted light was detected at 510 nm. Fura 2 fluorescence was calibrated according to the method described by Grynkiewicz et al.32
A model of perfused rabbit lungs has previously been described in detail (see overview33). Briefly, rabbits were ventilated with room air by use of a Harvard respirator (Hugo Sachs Elektronik). Catheters were placed into the pulmonary artery and left atrium, and the lungs were perfused with Krebs-Henseleit buffer. In parallel, room air ventilation was supplemented with 5% CO2. Lungs included in the present study had a homogeneous white appearance with no signs of hemostasis, edema, or atelectasis, and they had pulmonary artery and ventilation pressures in the normal range and were isogravimetric during an initial steady-state period of at least 30 minutes.
Capillary filtration coefficient (Kfc, normalized for wet lung weight) and total vascular compliance were determined gravimetrically, and lung weight gain was calculated. Lungs were perfused for 120 minutes in the absence or presence of 10−7 mol/L ADM. Perfusate (100 μmol H2O2 · min−1 · 150 mL−1) was infused as indicated. In all experiments, 5 μmol/L of thromboxane receptor antagonist BM 13.505 was admixed with the recirculating buffer fluid 10 minutes before stimulus application. Perfusate samples for determination of cAMP were taken after ADM stimulation and were processed for ELISA (Biotrend).
Depending on the number of groups (A) and the number of different time points studied (B), data of Figures 1, 2, and 6⇓⇓ were analyzed by an A×B ANOVA. A one-way ANOVA was used for data of Figures 3 and 4⇓A. Main effects were then compared by an F probability test. A value of P<0.05 was considered significant. Data are displayed as mean±SEM.
An expanded Materials and Methods section can be found in an online data supplement available at http://www.circresaha.org.
ADM Improves Barrier Function of Cultured Endothelial Cell Monolayers
Sealed PAEC monolayers (Figure 1) and HUVEC monolayers (Figure 2) displayed a hydraulic conductivity of <0.5×105 cm · s−1 · cm H2O−1. The addition of 1 μmol/L ADM as a bolus to PAEC (Figure 1) or HUVEC (Figure 2) monolayers and continuous infusion of 10 μmol/L ADM had no effect on the endothelial barrier function of resting cells within the time frame tested (90 minutes, data not shown).
First, we used H2O2 to mimic a polymorphonuclear leukocyte–mediated oxidant attack (Figures 1 and 2⇑A). H2O2 (1 mmol/L) time-dependently increased the endothelial monolayer permeability of PAECs (Figure 1) and HUVECs (Figure 2A). Pretreatment of PAECs with 0.01 to 1 μmol/L ADM or of HUVEC with 0.1 to 1 μmol/L ADM 15 minutes before the stimulus dose-dependently reduced the H2O2-related increase in endothelial hydraulic conductivity (Figures 1 and 2⇑A). Control monolayers were stable throughout the experimental period and responded promptly to the addition of staphylococcal α-toxin, a well-established permeabilizing agent, as shown for HUVECs (Figure 2A).
Second, HlyA was used as a prototype for a pore-forming bacterial toxin (Figure 2B). HlyA (0.1 hemolytic units [HU]) given as a bolus induced a rapid and strong increase in the hydraulic conductivity of HUVEC monolayers. Incubation of HUVECs with 0.1 to 1 μmol/L ADM 15 minutes before the addition of HlyA reduced the toxin-related increase in permeability (Figure 2B).
Third, we analyzed the effect of ADM on thrombin-mediated endothelial hyperpermeability (Figure 2C). Exposure of endothelial cell monolayers to 0.1 U thrombin resulted in a loss of barrier function within 20 minutes. The addition of 0.01 to 1 μmol/L ADM before thrombin stimulation substantially reduced the thrombin-induced hyperpermeability (Figure 2C). We then examined whether the inhibition of cAMP-specific PDE3/4 in combination with cAMP-elevating ADM had an effect on thrombin-mediated hyperpermeability (Figure 2D). Exposures of endothelial cell monolayers to concentrations of ADM (1 nmol/L, 15 minutes) and the PDE 3/4 inhibitor zardaverine (10 μmol/L, 30 minutes), which alone had no significant effect on thrombin-related hyperpermeability, were very effective when used in combination (Figure 2D).
ADM Binds to Endothelial Cells and Induces Formation of cAMP but Does Not Change cGMP or [Ca2+]i Levels
Human 125I-ADM binds to confluent HUVEC monolayers with a Bmax of 0.4 pmol per well and displays a Kd of ≈147.9 nmol/L. The specificity of ADM binding to HUVECs was confirmed by competition experiments using increasing concentrations of unlabeled ADM. Confluent PAEC cultures showed a Bmax of 0.25 pmol per well and a Kd of 13.4 nmol/L for human 125I-ADM, which could be blocked by increasing concentrations of unlabeled ADM, indicating specificity of peptide binding (see online data supplement).
We also confirmed the ability of ADM to stimulate cAMP formation in endothelial cells. Treatment of HUVECs or PAECs for 10 minutes with increasing concentrations of ADM (0.1 nmol/L to 10 μmol/L) induced cAMP formation in HUVECs (Figure 3A; data for PAECs are not shown) to a lesser extent than exposure to the positive control forskolin (1 μmol/L) for 10 minutes. Previous studies had shown high activities of PDE isoenzymes 2 to 4 in endothelial cells and a multiplying effect of specific PDE inhibition on cyclic nucleotide accumulation.8,9⇓ To determine the effect of PDE inhibition on ADM-stimulated cAMP accumulation, cells were pretreated with 10 μmol/L of the dual-selective PDE3/4 inhibitor zardaverine for 30 minutes, which resulted in a dramatic rise of ADM- or forskolin-mediated cAMP accumulation (Figure 3A). In contrast, cGMP accumulation in HUVECs (Figure 3B) or PAECs (data not shown) was unaffected after ADM stimulation (up to 100 μmol/L ADM for 5, 15, or 30 minutes) even in the presence of erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) (10 μmol/L), a PDE2 inhibitor (Figure 3B; data for 5 and 30 minutes are not shown). However, in the same experimental setup, a 4-fold increase of cGMP could be demonstrated when combining the PDE2 inhibitor EHNA with the NO donor sodium nitroprusside (10 μmol/L, 15 minutes) (Figure 3B).
An increase in [Ca2+]i induced by thrombin, H2O2, or HlyA treatment of endothelial cells was considered to be a strong signal for endothelial cell retraction followed by endothelial hyperpermeability. ADM exposure of HUVECs (1 to 100 μmol/L) had no effect on basal [Ca2+]i content within an observation period of 15 minutes (data not shown). We tested the hypothesis that ADM may reduce the H2O2-, thrombin-, or HlyA-mediated rise in [Ca2+]i, thereby preventing F-actin rearrangement and hyperpermeability. However, pretreatment of HUVECs with 10 μmol/L ADM before stimulation with H2O2, thrombin, or HlyA showed no effect on the [Ca2+]i increase induced by these particular agents (see online data supplement).
ADM Reduces Stimulus-Induced Phosphorylation of MLCs and Alterations of Human Endothelial Cell Filamentous Actin
Phosphorylation of regulatory MLC contributes significantly to endothelial cell contraction provoked by proinflammatory agents, thereby allowing paracellular fluid flux. Using an antibody directed against phospho-Thr18/phospho-Ser19 of MLC, we demonstrated that ADM reduced thrombin- and H2O2-dependent MLC phosphorylation (Figures 4A and 4B). Simultaneous detection of ERK1 confirmed equal protein loading.
In addition, ADM blocked H2O2-, thrombin-, or HlyA-mediated alterations of endothelial filamentous actin (F-actin), as shown by fluorescence microscopy (Figure 5). HUVECs exposed to solvent (Figure 5A) or 1 μmol/L ADM (Figure 5B) displayed a well-organized peripheral dense band of F-actin with only a few stress fibers. After stimulation of endothelial cells with 1 mmol/L H2O2 for 30 minutes (Figure 5C), 1 U thrombin for 15 minutes (Figure 5E), or 0.1 HU HlyA for 15 minutes (Figure 5G), the amount and density of stress fibers increased, whereas the peripheral dense band was disrupted, and gaps between the endothelial cells were opened. In contrast, in cells pretreated with 1 μmol/L ADM 15 minutes before stimulation with these agents (Figures 5D, 5F, and 5H), F-actin distribution remained unchanged, and intercellular gaps were closed as in the control monolayers.
Human ADM Stimulates cAMP Formation and Reduces H2O2-Induced Vascular Hyperpermeability in Isolated Rabbit Lungs
We made use of isolated, ventilated, blood-free perfused rabbit lungs stimulated with H2O2 (100 μmol/min admixed to the 150 mL perfusate over 15 minutes) to analyze the power of ADM in the regulation of endothelial barrier function in a more integrated model (Figure 6). In lungs exposed to H2O2 alone, Kfc increased up to 9.0±2.08 within 45 minutes, followed by massive pulmonary edema formation (Figure 6A). In contrast, ADM (0.1 μmol/L ADM 15 minutes before H2O2 admixture) almost completely prevented H2O2-induced edema formation (Figure 6A). The vascular compliance remained unchanged and was not different between the experimental groups. Pulmonary artery pressure showed some minor elevation on H2O2 infusion but did not display a significant difference between ADM-treated and -untreated lungs at the time points of the Kfc determination (see online data supplement). Moreover, only negligible changes (≤1 mm Hg) of microvascular pressures were observed in both experimental groups, indicating that the reduced edema formation observed in the ADM-stimulated rabbit lungs was not due to reduced filtration pressure (see online data supplement).
cAMP content in the perfusate collected from the rabbit lungs processed for analysis of Kfc was strongly increased in ADM-exposed lungs (Figure 6B).
Recent studies have demonstrated elevated plasma levels of ADM in vertebrates with a systemic inflammatory response.15–19⇓⇓⇓⇓ However, the role of ADM in the complex and dynamic disease process of sepsis is still largely undefined. On the one hand, the high ADM plasma levels observed in septic humans may contribute to hypotension and hyperdynamic circulatory response in sepsis, thereby contributing to disease progress.18 On the other hand, transgenic mice overexpressing ADM in their vasculature turned out to be resistant against lipopolysaccharide-induced shock, suggesting a rather beneficial effect of elevated ADM levels in sepsis.26
Considering that endothelial hyperpermeability is the hallmark of an inflammatory reaction1,2⇓ and that mice lacking a functional ADM gene displayed an extreme hydrops fetalis,27 we tested the hypothesis that elevated ADM levels stabilized endothelial barrier function, thereby acting as a “protective” peptide in the systemic inflammatory response. Our results clearly indicate that ADM potently stabilized endothelial barrier function. ADM preincubation reduced endothelial hyperpermeability induced by thrombin, H2O2, or HlyA in vitro. Moreover, in isolated perfused rabbit lungs, ADM decreased H2O2-related capillary hyperpermeability and edema formation. Specific binding of ADM to endothelial cells elevated intracellular cAMP and reduced MLC phosphorylation, thereby stabilizing the endothelial cell microfilament system.
Because a broad variety of stimuli may contribute to endothelial barrier dysfunction in sepsis, we chose three typical permeability-inducing agents to investigate the effects of ADM. H2O2, released by polymorphonuclear granulocytes in inflammatory reactions, activates complex signaling pathways in endothelial cells, including active myosin-based cell contraction2 and protein kinase C (PKC) activation.7 HlyA, as a prototype of a bacterial pore-forming exotoxin, allows Ca2+ influx into the cytosol according to the transmembrane Ca2+ gradient and potently induces NO production13 as well as the expression of endothelial cell adhesion molecules.29 Thrombin exposure of endothelial cells activates phospholipid hydrolysis, increases [Ca2+]i, and promotes cell contraction.2,5,11⇓⇓
All three stimuli induced active actin-myosin–based cell contraction, thereby increasing enhanced paracellular endothelial permeability. Notably, ADM preexposure of endothelial cell monolayers greatly reduced the increase in hydraulic conductivity in response to all three agents. It has been suggested that ADM-mediated cAMP elevation contributes to ADM effects in the vasculature.19,24,25⇓⇓ In line with previous studies,20,21,24,25⇓⇓⇓ ADM incubation increased cAMP content in cultured endothelial cells. Human and porcine endothelial cells contain PDE3 and PDE4 for the degradation of cAMP8 and PDE2 for the degradation of cGMP.9 Inhibition of PDE3/4 with the dual-selective PDE3/4 inhibitor zardaverine significantly increased ADM-related cAMP accumulation and strengthened the endothelial barrier–protective effect of ADM as assessed for thrombin-related hyperpermeability. ADM seems to be as potent as a pharmacological stimulator of cAMP elevation, regarding maintenance of endothelial barrier function.8,9⇓ Moreover, ADM treatment elevated cAMP levels in isolated perfused rabbit lungs and reduced H2O2-related edema formation. This is consistent with previous studies showing that cAMP elevation potently blocks hyperpermeability in isolated rabbit lungs.34
To exclude the effects of H2O2-dependent pulmonary vasoconstriction on edema formation, we used the thromboxane receptor antagonist BM 13.505. Although ADM was known to reduce systemic blood pressure15,35⇓ and, in some systems, pulmonary hypertension,36 no substantial changes in pulmonary artery perfusion pressure were noted in H2O2- or solvent-exposed rabbit lungs pretreated with BM 13.505.
In line with the presently described role of ADM in the regulation of endothelial permeability under inflammatory conditions, recent observations in mice lacking a functional ADM gene point to a general role of ADM in permeability regulation: mice lacking a functional ADM gene displayed an extreme hydrops fetalis as well as cardiovascular abnormalities and died at mid gestation.27 Overall, these observations suggest a pivotal role of ADM in the regulation of endothelial barrier function.
Alterations of the endothelial cell microfilament system are accompanied by active actin-myosin–based cell contraction, allowing increased paracellular fluid flux, which seems to be critical for edema formation under inflammatory conditions.1,2,4,5,8,9,11⇓⇓⇓⇓⇓⇓ ADM reduced thrombin- and H2O2-related phosphorylation of MLC and blocked endothelial cell contraction, intercellular gap formation, and stress fiber formation. Besides classic Ca2+/calmodulin-dependent MLC kinases, 3 Rho kinase may contribute to thrombin-related MLC phosphorylation. Although MLC kinase phosphorylates MLC at Ser19 and Thr18 as a sole mode of action, Rho kinase additionally blocks myosin phosphatase type 1, thereby enhancing MLC phosphorylation.4,5,37⇓⇓ Inasmuch as cAMP elevation reduced Rho kinase–dependent phosphorylation of MLC in lipopolysaccharide-exposed endothelial cells38 and Rho kinase was identified as a central regulator of thrombin-induced endothelial cell contraction,4,5,37⇓⇓ ADM-related increased cAMP may act via inhibition of the Rho–Rho kinase pathway. Moreover, PKC-dependent phosphorylations of MLC and important permeability-regulating junctional proteins, such as vasodilator-stimulated phosphoprotein,39 zonula occludens protein-1,2,39⇓ and vascular endothelial cadherin,2 significantly contribute to barrier dysfunction. Although cAMP elevation seems not to prevent stimulus-dependent PKC activation in endothelial cells,40 cAMP-related PKA activation may counterregulate PKC-induced phosphorylation effects. For example, it has been shown that PKA-dependent phosphorylation of the Ser157 residue of vasodilator-stimulated phosphoprotein diminishes paracellular permeability through the relaxation of actin cytoskeletal tension.39
The data presented support the notion that ADM binds via specific receptors to endothelial cells. However, the situation is complex inasmuch as CRLR and RAMP2 and RAMP3 together form ADM receptors.20,21,23⇓⇓ Moreover, alternative ADM binding sites may exist. Interestingly, the expression of CRLR-RAMP2/CRLR-RAMP3 complexes apparently undergoes regulatory changes in different tissues and stages during sepsis, thereby contributing to ADM-related effects in systemic inflammation.41
Besides the widely accepted ADM-dependent cAMP elevation, conflicting results were reported regarding an ADM-induced rise in [Ca2+]i in endothelial cells.20,24,25⇓⇓ In the present study, ADM exposure had no effect on [Ca2+]i levels in cultured HUVECs and showed no modulation of the H2O2-, HlyA-, or thrombin-mediated increase in [Ca2+]i. In addition, ADM-treated endothelial cells displayed no changes of intracellular cGMP content, even in cells with PDE2 inhibition to block cGMP degradation.
In summary, the data presented indicate that specific binding of ADM to endothelial cells elevated cAMP levels, blocked H2O2- and thrombin-related MLC-phosphorylation, and prevented endothelial cell contraction. ADM markedly reduced thrombin-, HlyA-, and H2O2-related endothelial hyperpermeability. Simultaneous inhibition of cAMP-degrading PDE3/4 and ADM treatment acted synergistically. Moreover, treatment of rabbit lungs with ADM reduced H2O2-induced edema formation and increased cAMP levels in lung perfusate. Thus, ADM has the potential of being a new therapeutic tool in systemic inflammatory reactions by stabilizing the endothelial barrier and preventing vascular leakage.
This work was supported by the Deutsche Forschungsgemeinschaft (SFB 547/C2 to Dr Rascher and SFB 547/B6 to Dr Schütte) and the German Federal Research Ministry (BMBF) to Dr Suttorp (CAPNETZ/C4). Parts of this work will be included in the MD thesis of M. Krisp.
Original received January 11, 2002; resubmission received July 26, 2002; accepted August 28, 2002.
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