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Only some subpopulations of natural killer (NK) cells have cytotoxic functionality, and the effects of anesthetics on these subpopulations are unknown. This study aimed to evaluate the in vitro effects of various anesthetics, both alone and in combination, on the distribution and cytotoxic function of NK cells and their subpopulations. Peripheral blood mononuclear cells (PBMCs) from eight healthy volunteers were treated for 4 h in vitro with dexmedetomidine, remifentanil, lidocaine, propofol, sevoflurane, and combinations in clinically relevant concentrations or left untreated. Flow cytometry was used to quantify the percentage of sampled NK cells and evaluate their distribution (CD56 bright CD16 neg , CD56 bright CD16 dim , CD56 dim CD16 neg , CD56 dim CD16 bright , and CD56 neg CD16 bright ) and cytotoxicity (Granzyme B (GrzB) and perforin) of NK cell subpopulations. Although the percentage of total NK cells did not change following exposure to anesthesia, the most important cytotoxic subpopulation (CD56 dim CD16 bright NK cells) decreased after exposure to both propofol (-3.58%, p = 0.045) and sevoflurane (-16.10%, p = 0.008) alone, and most combinations, especially in combination with lidocaine (propofol with lidocaine (-9.66%, p = 0.002) and sevoflurane with lidocaine (-21.90%, p < 0.001)). Dexmedetomidine and remifentanil had no effect on CD56 dim CD16 bright NK cells. Furthermore, no anesthetic regimen or combination altered the expression of GrzB and perforin in NK cells or NK cell subpopulations. In short, propofol and sevoflurane suppressed the highly cytotoxic phenotype (CD56 dim CD16 bright ) of NK cells, with those exposed to sevoflurane combinations showing greater reductions. Immunosuppression was intensified with the inclusion of lidocaine in the anesthetic regimen., Competing Interests: Tristan J. Vulcano: No conflicts of interest to report. Wayel H. Abdulahad: No conflicts of interest to report. Matijs van Meurs: No conflicts of interest to report. Rianne M. Jongman: No conflicts of interest to report. Michel M.R.F. Struys: The author has no conflicts of interest related to this manuscript. In general, his research group/department received (over the last 3 years) research grants and consultancy fees from Masimo (Irvine, CA, USA), Becton Dickinson (Eysins, Switzerland), Fresenius (Bad Homburg, Germany), Paion (Aachen, Germany), Medcaptain Europe (Andelst, The Netherlands), Baxter (Chicago, Il, USA), and HanaPharm (Seoul, Republic of Korea). He receives royalties on intellectual property from Demed Medical (Sinaai, Belgium) and Ghent University (Gent, Belgium). Dirk J. Bosch: No conflicts of interest to report.

Introduction: Targeted temperature management (TTM) is considered to be a neuroprotective strategy during cardiopulmonary bypass (CPB) assisted procedures, possibly through the activation of cold shock proteins. We therefore investigated the effects of mild compared with deep hypothermia on the neuroinflammatory response and cold shock protein expression after CPB in rats., Methods: Wistar rats were subjected to 1 hr of mild (33 °C) or deep (18 °C) hypothermia during CPB or sham procedure. PET scan analyses using TSPO ligand [ 11 C]PBR28 were performed on day 1 (short-term) or day 3 and 7 post-procedure (long-term) to assess neuroinflammation. Hippocampal and cortical samples were obtained at day 1 in the short-term group and at day 7 in the long-term group. mRNA expression of M1 and M2 microglia associated cytokines was analysed with RT-PCR. Cold shock protein RNA-binding motive 3 (RBM3) and tyrosine receptor kinase B (TrkB) receptor protein expression were determined with Western Blot and quantified., Results: In both groups target temperature was reached within an hour. Standard uptake values (SUV) of [ 11 C]PBR28 in CPB rats at 1 day and 3 days were similar to that of sham animals. At 7 days after CPB the SUV was significantly higher in amygdala and hippocampal regions of the CPB 18 °C group as compared to the CPB 33 °C group. No differences were observed in the expression of M1 and M2 microglia-related cytokines between TTM 18 °C and 33 °C. RBM3 protein levels in cortex and hippocampus were significantly higher in CPB 33 °C compared to CPB 18 °C and sham 33 °C, at day 1 and day 7, respectively., Conclusions: TTM at 18 °C increased the neuroinflammatory response in amygdala and hippocampus compared to TTM at 33 °C in rats undergoing a CPB procedure. Additionally, TTM at 33 °C induced increased expression of TrkB and RBM3 in cortex and hippocampus of rats on CPB compared to TTM at 18 °C. Together, these data indicate that neuroinflammation is alleviated by TTM at 33 °C, possibly by recruiting protective mechanisms through cold shock protein induction., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

Background: Acute kidney injury (AKI) in sepsis patients increases patient mortality. Endothelial cells are important players in the pathophysiology of sepsis-associated AKI (SA-AKI), yet knowledge regarding their spatiotemporal involvement in coagulation disbalance and leukocyte recruitment is lacking. This study investigated the identity and kinetics of responses of different microvascular compartments in kidney cortex in response to SA-AKI., Methods: Laser microdissected arterioles, glomeruli, peritubular capillaries, and postcapillary venules from kidneys of mice subjected to cecal ligation and puncture (CLP) were analyzed using RNA sequencing. Differential expression and pathway enrichment analyses identified genes involved in coagulation and inflammation. A selection of these genes was evaluated by RT-qPCR in microvascular compartments of renal biopsies from patients with SA-AKI. The role of two identified genes in lipopolysaccharide-induced endothelial coagulation and inflammatory activation were determined in vitro in HUVEC using siRNA-based gene silencing., Results: CLP-sepsis in mice induced altered expression of approximately 400 genes in the renal microvasculature, with microvascular compartments exhibiting unique spatiotemporal responses. In mice, changes in gene expression related to coagulation and inflammation were most extensive in glomeruli at early and intermediate time points, with high induction of Plat, Serpine1, Thbd, Icam1, Stat3, and Ifitm3. In human SA-AKI, PROCR and STAT3 were induced in postcapillary venules, while SERPINE1 expression was diminished. IFITM3 was increased in arterioles and glomeruli. In vitro studies revealed that STAT3 and IFITM3 partly control endothelial coagulation and inflammatory activation., Conclusion: Renal microvascular compartments in mice and humans exhibited heterogeneous changes in coagulation- and inflammation-related gene expression in response to SA-AKI. Additional research should aim at understanding the functional consequences of the here described heterogeneous microvascular responses to establish the usefulness of identified genes as therapeutic targets in SA-AKI., Competing Interests: Declaration of competing interest M. Hackl is co-founder and CEO/CSO of TAmiRNA GmbH, which provides small RNA sequencing and RNA sequencing analysis services. A.B. Diendorfer, M. Pultar, and S. Skalicky are employed by TAmiRNA. G. Molema is co-founder and CSO/CTO of Vivomicx BV, which provides laser microdissection analysis services. Vivomicx and TAmiRNA financially contributed to the PPP allowance. The remaining authors declare no conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Endothelial cells in blood vessels in the kidney exert different functions depending on the (micro)vascular bed they are located in. The present study aimed to investigate microRNA and mRNA transcription patterns that underlie these differences. We zoomed in on microvascular compartments in the mouse renal cortex by laser microdissecting the microvessels prior to small RNA- and RNA-sequencing analyses. By these means, we characterized microRNA and mRNA transcription profiles of arterioles, glomeruli, peritubular capillaries, and postcapillary venules. Quantitative RT-PCR, in situ hybridization, and immunohistochemistry were used to validate sequencing results. Unique microRNA and mRNA transcription profiles were found in all microvascular compartments, with dedicated marker microRNAs and mRNAs showing enriched transcription in a single microvascular compartment. In situ hybridization validated the localization of microRNAs mmu-miR-140-3p in arterioles, mmu-miR-322-3p in glomeruli, and mmu-miR-451a in postcapillary venules. Immunohistochemical staining showed that von Willebrand factor protein was mainly expressed in arterioles and postcapillary venules, whereas GABRB1 expression was enriched in glomeruli, and IGF1 was enriched in postcapillary venules. More than 550 compartment-specific microRNA-mRNA interaction pairs were identified that carry functional implications for microvascular behavior. In conclusion, our study identified unique microRNA and mRNA transcription patterns in microvascular compartments of the mouse kidney cortex that underlie microvascular heterogeneity. These patterns provide important molecular information for future studies into differential microvascular engagement in health and disease. NEW & NOTEWORTHY Renal endothelial cells display a high level of heterogeneity depending on the (micro)vascular bed they reside in. The molecular basis contributing to these differences is poorly understood yet of high importance to increase understanding of microvascular engagement in the kidney in health and disease. This report describes m(icro)RNA expression profiles of microvascular beds in the mouse renal cortex and uncovers microvascular compartment-specific m(icro)RNAs and miRNA-mRNA pairs, thereby revealing important molecular mechanisms underlying renal microvascular heterogeneity.

Endothelial cells (ECs) in the microvasculature in organs are active participants in the pathophysiology of sepsis. Tyrosine protein kinase receptor Tie2 (Tek; Tunica interna Endothelial cell Kinase) is thought to play a role in their inflammatory response, yet data are inconclusive. We investigated acute endotoxemia-induced changes in the expression of Tie2 and inflammation-associated endothelial adhesion molecules E-selectin and VCAM-1 (vascular cell adhesion molecule-1) in kidneys and lungs in inducible, EC-specific Tie2 knockout mice. The extent of Tie2 knockout in healthy mice differed between microvascular beds, with low to absent expression in arterioles in kidneys and in capillaries in lungs. In kidneys, Tie2 mRNA dropped more than 70% upon challenge with lipopolysaccharide (LPS) in both genotypes, with no change in protein. In renal arterioles, tamoxifen-induced Tie2 knockout was associated with higher VCAM-1 protein expression in healthy conditions. This did not increase further upon challenge of mice with LPS, in contrast to the increased expression occurring in control mice. Also, in lungs, Tie2 mRNA levels dropped within 4 h after LPS challenge in both genotypes, while Tie2 protein levels did not change. In alveolar capillaries, where tamoxifen-induced Tie2 knockout did not affect the basal expression of either adhesion molecule, a 4-fold higher E-selectin protein expression was observed after exposure to LPS compared to controls. The here-revealed heterogeneous effects of absence of Tie2 in ECs in kidney and lung microvasculature in health and in response to acute inflammatory activation calls for further in vivo investigations into the role of Tie2 in EC behavior.

Major surgery induces systemic inflammation leading to pro-inflammatory activation of endothelial cells. Endothelial inflammation is one of the drivers of postoperative organ damage, including acute kidney injury Tumour Necrosis Factor alpha (TNF-α) is an important component of surgery-induced pro-inflammatory activation of endothelial cells. Kinases, the backbone of signalling cascades, can be targeted by pharmacological inhibition. This is a promising treatment option to interfere with excessive endothelial inflammation. In this study, we identified activated kinases as potential therapeutic targets. These targets were pharmacologically inhibited to reduce TNF-α-induced pro-inflammatory signalling in endothelial cells. Kinome profiling using PamChip arrays identified 64 protein tyrosine kinases and 88 serine-threonine kinases, the activity of which was determined at various timepoints (5-240 min) following stimulation with 10 ng/ml TNF-α in Human umbilical vein endothelial cells in vitro . The PTKs Axl and Fyn were selected based on high kinase activity profiles. Co-localisation experiments with the endothelial-specific protein CD31 showed Axl expression in endothelial cells of glomeruli and Fyn in arterioles and glomeruli of both control and TNF-α-exposed mice. Pharmacological inhibition with Axl inhibitor BMS-777607 and Fyn inhibitor PP2 significantly reduced TNF-α-induced pro-inflammatory activation of E-selectin, VCAM-1, ICAM-1, IL-6 and IL-8 at mRNA and VCAM-1, ICAM-1, and IL-6 at protein level in HUVEC in vitro . Upon pharmacological inhibition with each inhibitor, leukocyte adhesion to HUVEC was also significantly reduced, however to a minor extent. In conclusion, pre-treatment of endothelial cells with kinase inhibitors BMS-777607 and PP2 reduces TNF-α-induced endothelial inflammation in vitro ., Competing Interests: TS is currently working as Senior Medical Director for Edwards Lifesciences (Unterschleißheim, Germany). The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Ellermann, Jongman, Luxen, Kuiper, Plantinga, Moser, Scheeren, Theilmeier, Molema and Van Meurs.)

Tyrosine-protein kinase receptor Tie2, also known as Tunica interna Endothelial cell Kinase or TEK plays a prominent role in endothelial responses to angiogenic and inflammatory stimuli. Here we generated a novel inducible Tie2 knockout mouse model, which targets mature (micro)vascular endothelium, enabling the study of the organ-specific contribution of Tie2 to these responses. Mice with floxed Tie2 exon 9 alleles (Tie2floxed/floxed) were crossed with end-SCL-Cre-ERT transgenic mice, generating offspring in which Tie2 exon 9 is deleted in the endothelial compartment upon tamoxifen-induced activation of Cre-recombinase (Tie2ΔE9). Successful deletion of Tie2 exon 9 in kidney, lung, heart, aorta, and liver, was accompanied by a heterogeneous, organ-dependent reduction in Tie2 mRNA and protein expression. Microvascular compartment-specific reduction in Tie2 mRNA and protein occurred in arterioles of all studied organs, in renal glomeruli, and in lung capillaries. In kidney, lung, and heart, reduced Tie2 expression was accompanied by a reduction in Tie1 mRNA expression. The heterogeneous, organ- and microvascular compartment-dependent knockout pattern of Tie2 in the Tie2floxed/floxed;end-SCL-Cre-ERT mouse model suggests that future studies using similar knockout strategies should include a meticulous analysis of the knockout extent of the gene of interest, prior to studying its role in pathological conditions, so that proper conclusions can be drawn., Competing Interests: The authors have declared that no competing interest exist.

Background: Evidence shows that activation of pulmonary vascular endothelium and neutrophils are involved in the pathophysiology of acute bronchiolitis. We hypothesized that levels of markers of endothelial activation and leukocyte counts are associated with requirement and duration of respiratory support., Methods: Thirty-four infants with bronchiolitis and eight controls were included. Nasopharyngeal swabs and blood samples were taken at admission. Serum levels of Angiopoietin (Ang)-1, Ang-2, sP-selectin, sE-selectin, vascular cell adhesion molecule-1 (sVCAM-1), intercellular adhesion molecule-1 (sICAM-1), and leukocyte counts were measured. For univariate analysis, bronchiolitis cases were grouped into two groups, namely those not requiring and those requiring any form of respiratory support. To control for known risk factors for poor outcome (i.e., age, prematurity, and congenital heart disease), and for days post symptom onset, linear regression analysis was performed with duration of any type of respiratory support in days., Results: Ang-2 levels, Ang-2/Ang-1 ratios, sE-selectin levels, immature neutrophil count, and neutrophil/lymphocyte ratio (NLR) were higher in acute bronchiolitis versus controls. Ang-2, and NLR levels were significantly higher, and lymphocyte counts significantly lower, in infants that required respiratory support versus those that did not. Ang-2 levels (β: .32, 95% confidence interval [CI]: 0.19-1.19) and NLR (β: .68, 95% CI: 0.17-1.19) were positive predictors for the duration of respiratory support., Conclusions: Markers of endothelial and neutrophil activation are associated with respiratory support for acute bronchiolitis. Admission Ang-2 levels and NLR may be promising markers to determine requirement of respiratory support and deserve further study., (© 2021 Wiley Periodicals LLC.)

Background: Cardiac surgery with cardiopulmonary bypass (CPB) is commonly associated with acute kidney injury, and microvascular endothelial inflammation is a potential underlying mechanism. We hypothesized that pro-inflammatory components of plasma from patients who underwent coronary artery bypass graft surgery with CPB induce endothelial adhesion molecule expression when incorporating altered shear stress in the in vitro model., Methods: The clinical characteristics and markers of systemic inflammation and kidney injury were analyzed pre and postoperatively in 29 patients undergoing coronary artery bypass grafting with CPB. The effects of tumor necrosis factor (TNF)-α and patient plasma on the expression of endothelial inflammation and adhesion markers were analyzed in vitro ., Results: Plasma TNF-α was elevated 6 h postoperation (median: 7.3 pg/ml (range: 2.5-94.8 pg/ml)). Neutrophil gelatinase-associated lipocalin in plasma peaked 6 h (99.8 ng/ml (52.6-359.1 ng/ml)) and in urine 24 h postoperation (1.6 ng/mg (0.2-6.4 ng/mg)). Urinary kidney injury molecule-1 concentration peaked 24 h postoperation (0.5 ng/mg (0.2-1.2 ng/mg). In vitro , the expression of E-selectin was induced by 20 pg/ml TNF-α. In addition, the expression of interleukin-8, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1 was induced by 100 pg/ml TNF-α. Compared to healthy control plasma exposure, postoperative plasma did not increase the expression of markers of endothelial inflammation and adhesion under shear stress in vitro ., Conclusion: Patients undergoing CPB surgery showed mild systemic inflammation and kidney injury. However, the plasma components did not stimulate endothelial inflammation and adhesion molecule expression in vitro ., Competing Interests: There are no conflicts of interest., (Copyright: © 2021 International Journal of Critical Illness and Injury Science.)

Background: Anaesthetic agents are likely to alter circulating cytokine concentrations. Because preceding studies have not been able to exclude the contribution of surgical trauma, perioperative stress, or both to circulating cytokine concentrations, the effects of anaesthesia remain unclear. The aim of this study was to quantify serum cytokines in healthy volunteers administered i.v. anaesthetic agents in the absence of surgical trauma and perioperative stress., Methods: Serum samples obtained during previous standardised studies from healthy volunteers were compared before and 6-8 h after induction of anaesthesia with propofol (n=31), propofol/remifentanil (n=30), dexmedetomidine (n=17) or dexmedetomidine/remifentanil (n=15). Anaesthetic regimens were standardised and volunteers did not undergo any surgical intervention. Serum concentrations of interleukin (IL)2, IL4, IL6, IL10, IL17, IL18, IL21, IL22, IL23, C-X-C motif ligand 8, interferon gamma, E-selectin, L-selectin, major histocompatibility complex class I chain-polypeptide-related sequence (MIC)A, MICB, Granzyme A, and Granzyme B were quantified using a multiplexed antibody-based assay (Luminex)., Results: Samples were obtained from volunteers of either sex aged 18-70 yr. After anaesthesia with propofol alone, concentrations of IL4 (P=0.012), IL6 (P=0.027), IL21 (P=0.035), IL22 (P=0.002), C-X-C motif ligand 8 (P=0.004), MICB (P=0.046), and Granzyme A (P=0.045) increased. After anaesthesia with propofol and remifentanil, IL17 (P=0.013), interferon gamma (P=0.003), and MICA (P=0.001) decreased, but IL6 (P=0.006) and L-selectin (P=0.001) increased. After dexmedetomidine alone, IL18 (P=0.002), L-selectin (P=0.017), E-selectin (P=0.002), and Granzyme B (P=0.023) decreased. After dexmedetomidine with remifentanil no changes were observed., Conclusions: In healthy volunteers not undergoing surgery, different i.v. anaesthesia regimens were associated with differential effects on circulating cytokines., (Copyright © 2020 British Journal of Anaesthesia. Published by Elsevier Ltd. All rights reserved.)

Background: Serum levels of markers of endothelial cell activation are associated with bacteremia and mortality in sepsis in adults, children, and newborns with early onset sepsis. We hypothesize that levels of these markers are associated with these outcomes in hospitalized newborns with suspected late onset neonatal sepsis (LONS)., Methods: In this prospective cohort study, newborns admitted to the tertiary neonatal care facility of Suriname were included upon clinical suspicion of LONS and before start of antibiotic treatment, between April 1, 2015 and May 31, 2016. Serum concentrations of angiopoietin-1, angiopoietin-2, and soluble isoforms of P-selectin, E-selectin, vascular cell adhesion molecule-1 (sVCAM-1), intercellular adhesion molecule-1 (sICAM-1), platelet and endothelial cell adhesion molecule-1 (sPECAM-1), matrix metalloproteinase-9 (MMP-9), neutrophil elastase, and tissue-inhibitor of metalloproteinases-1 (TIMP-1) were measured., Results: Twenty-thee newborns were included. Baseline characteristics were similar between newborns with and without bacteremia and between non-survivors and survivors. Only soluble E-selectin (sE-selectin) was higher in newborns with bacteremia versus non-bacteremia (P=0.04) and lower in non-survivors (P=0.04). No conclusions could be made for sVCAM-1 due to high serum concentrations., Conclusions: In conclusion, the data from this pilot study indicate that serum levels of markers of endothelial cell activation are poorly associated with bacteremia and mortality., Competing Interests: Conflicts of Interest: The authors have no conflicts of interest to declare., (2019 Translational Pediatrics. All rights reserved.)

Tyrosine kinase receptor (Tie2) is mainly expressed by endothelial cells. In animal models mimicking critical illness, Tie2 levels in organs are temporarily reduced. Functional consequences of these reduced Tie2 levels on microvascular endothelial behavior are unknown. We investigated the effect of partial deletion of Tie2 on the inflammatory status of endothelial cells in different organs. Newly generated heterozygous Tie2 knockout mice (exon 9 deletion, ΔE9/Tie2) exhibiting 50% reduction in Tie2 mRNA and protein, and wild-type littermate controls (Tie2), were subjected to hemorrhagic shock and resuscitation (HS + R), or challenged with i.p. lipopolysaccharide (LPS). Kidney, liver, lung, heart, brain, and intestine were analyzed for mRNA levels of adhesion molecules E-selectin, vascular cell adhesion molecule 1 (VCAM-1), and intercellular cell adhesion molecule 1 (ICAM-1), and CD45. Exposure to HS + R did not result in different expression responses of these molecules between organs from Tie2 or Tie2 mice and sham-operated mice. In contrast, the LPS-induced mRNA expression levels of E-selectin, VCAM-1, and ICAM-1, and CD45 in organs were attenuated in Tie2 mice when compared with Tie2 mice in kidney and liver, but not in the other organs studied. Furthermore, reduced expression of E-selectin and VCAM-1 protein, and reduced influx of CD45 cells upon LPS exposure, was visible in a microvascular bed-specific pattern in kidney and liver of Tie2 mice compared with controls. In contrast to the hypothesis that a disbalance in the Ang/Tie2 system leads to increased microvascular inflammation, heterozygous deletion of Tie2 is associated with an organ-restricted, microvascular bed-specific attenuation of endothelial inflammatory response to LPS.

Background: Early onset sepsis (EOS) is defined as onset of sepsis within 72 hours after birth. Leucocyte-endothelial interactions play a pivotal part in EOS pathophysiology. Endothelial cell adhesion molecules (CAMs) orchestrate these interactions and their soluble isoforms (sCAMs) are released into the vasculature by enzymes called sheddases., Purpose: This study was undertaken to explore further the pathophysiology of EOS and to investigate the potential of sCAM and their sheddases as potential biomarkers for EOS., Methods: Stored serum aliquots were used from 71 Surinamese newborns suspected of EOS and 20 healthy newborns from an earlier study. Serum had been collected within 72 hours after birth and six (8.6%) newborns had a positive blood culture with gram-negative pathogens. Concentrations of sCAMs sP-selectin, sE-selectin, soluble vascular cell adhesion molecule-1, intercellular adhesion molecule-1 and platelet and endothelial cell adhesion molecule-1, sheddases matrix metalloproteinase-9 (MMP-9) and neutrophil elastase (NE) and sheddase antagonist tissue-inhibitor of metalloproteinases-1 (TIMP-1) were measured simultaneously with Luminex and ELISA., Results: MMP-9 and TIMP-1 levels were measured in serum of n=91 newborns and sCAMs and NE levels in serum of n=80 newborns, respectively. We found no differences in median concentrations of sCAMs, MMP-9 and TIMP-1 or NE between blood culture positive EOS, blood culture negative EOS and control groups at start of antibiotic treatment., Conclusions: Our data indicate that serum concentrations of sCAMs and their sheddases have no clinical utility as biomarkers for EOS., Trial Registration Number: NCT02486783. Results., Competing Interests: Competing interests: None declared.

Background: Cardiopulmonary bypass during cardiac surgery leads to impaired microcirculatory perfusion. We hypothesized that vascular leakage is an important contributor to microcirculatory dysfunction. Imatinib, a tyrosine kinase inhibitor, has been shown to reduce vascular leakage in septic mice. We investigated whether prevention of vascular leakage using imatinib preserves microcirculatory perfusion and reduces organ injury markers in a rat model of cardiopulmonary bypass., Methods: Male Wistar rats underwent cardiopulmonary bypass after treatment with imatinib or vehicle (n=8 per group). Cremaster muscle microcirculatory perfusion and quadriceps microvascular oxygen saturation were measured using intravital microscopy and reflectance spectroscopy. Evans Blue extravasation was determined in separate experiments. Organ injury markers were determined in plasma, intestine, kidney, and lungs., Results: The onset of cardiopulmonary bypass decreased the number of perfused microvessels by 40% in the control group [9.4 (8.6-10.6) to 5.7 (4.8-6.2) per microscope field; P<0.001 vs baseline], whereas this reduction was not seen in the imatinib group. In the control group, the number of perfused capillaries remained low throughout the experiment, whilst perfusion remained normal after imatinib administration. Microvascular oxygen saturation was less impaired after imatinib treatment compared with controls. Imatinib reduced vascular leakage and decreased fluid resuscitation compared with control [3 (3-6) vs 12 ml (7-16); P=0.024]. Plasma neutrophil-gelatinase-associated-lipocalin concentrations were reduced by imatinib., Conclusions: Prevention of endothelial barrier dysfunction using imatinib preserved microcirculatory perfusion and oxygenation during and after cardiopulmonary bypass. Moreover, imatinib-induced protection of endothelial barrier integrity reduced fluid-resuscitation requirements and attenuated renal and pulmonary injury markers., (Copyright © 2017 British Journal of Anaesthesia. Published by Elsevier Ltd. All rights reserved.)

Microvascular endothelial cells play a pivotal role in the pathogenesis of sepsis-induced inflammatory responses and multiple organ failure. Therefore, they represent an important target for pharmacological intervention in the treatment of sepsis. Glucocorticosteroids were widely used in the treatment of sepsis but vast evidence to support their systemic use is lacking. The limited effects of glucocorticoids in the treatment of sepsis may be explained by differential effects of drug initiated NF-κB inhibition in different cell types and insufficient drug delivery in target cells. The current study aimed therefore to investigate the effects of an endothelial targeted delivery of dexamethasone in a mouse model of endotoxemia induced by two consecutive i.p. injections of lipopolysaccharide (LPS). To achieve endothelial cell specific delivery of dexamethasone, we modified SAINT-O-Somes, a new generation of liposomes that contain the cationic amphiphile SAINT-C18 (1-methyl-4-(cis-9-dioleyl) methyl-pyridinium chloride, with antibodies against vascular cell adhesion molecule-1 (VCAM-1). In LPS challenged mice, the systemic administration of free dexamethasone had negligible effects on the microvascular inflammatory endothelial responses. Dexamethasone-loaded anti-VCAM-1 SAINT-O-Somes specifically localized at VCAM-1 expressing endothelial cells in the microvasculature of inflamed organs. This was associated with a marginal attenuation of the expression of a few pro-inflammatory genes in kidney and liver, while no effects in the lung were observed. This study reveals that, although local accumulation of the targeted drug was achieved, endothelial targeted dexamethasone containing anti-VCAM-1 SAINT-O-Somes exhibited marginal effects on inflammatory endothelial cell activation in a model of endotoxemia. Studies with more potent drugs encapsulated into anti-VCAM-1 SAINT-O-Somes will in the future reveal whether this delivery system can be further developed for efficacious endothelial directed delivery of drugs in the treatment of sepsis.

In patients with sepsis-induced multi-organ dysfunction syndrome, diverging patterns of oedema formation and loss of function in organs such as lung and kidney suggest that endothelial permeability-regulating molecular responses are differentially regulated. This potential differential regulation has been insufficiently studied at the level of components of adherens and tight junctions. We hypothesized that such a regulation by endothelial cells in sepsis takes place in an organ-specific manner. We addressed our hypothesis by studying by quantitative real time polymerase chain reaction the expression of a predefined subset of EC permeability-related molecules (occludin, claudin-5, PV-1, CD-31, endomucin, Angiopoietin-1, Angiopoietin-2, Tie2, VEGFA, VEGFR1, VEGFR2, and VE-cadherin) in kidney and lung after systemic lipopolysacharide injection in mice, and in kidneys of patients who died of sepsis. We showed that baseline endothelial expression of permeability-related molecules differs in mouse kidney and lung. Moreover, we showed differential regulation of these molecules after lipopolysacharide injection in the two mouse organs. In lung we found a decrease in expression levels of molecules of the adherence and tight junctions complex and related signaling systems, compatible with increased permeability. In contrast, in kidney we found expression patterns of these molecules compatible with decreased permeability. Finally, we partially corroborated our findings in mouse kidney in human kidneys from septic patients. These findings may help to understand the clinical difference in the extent of oedema formation in kidney and lung in sepsis-associated organ failure.

Sepsis is a severe systemic inflammatory response to infection. Endothelial activation and dysfunction play a critical role in the pathophysiology of sepsis and represent an important therapeutic target to reduce sepsis mortality. Interferon regulatory factor 1 (IRF-1) was recently identified as a downstream target of TNF-α-mediated signal transduction in endothelial cells. The aim of this study was to explore the importance of IRF-1 as a regulator of lipopolysaccharide (LPS)-induced endothelial proinflammatory activation. We found that renal IRF-1 was upregulated by LPS in vivo as well as in LPS-stimulated endothelial cells in vitro. Furthermore, we identified intracellular retinoic acid inducible gene-I (RIG-I) as a regulator of LPS-mediated IRF-1 induction. IRF-1 depletion specifically resulted in diminished induction of VCAM-1 in response to LPS, but not of E-selectin or ICAM-1, which was independent of NFκB signaling. When both IRF-1 and the RIG-I adapter protein mitochondrial antiviral signaling (MAVS) were absent, VCAM-1 induction was not additionally inhibited, suggesting that MAVS and IRF-1 reside in the same signaling pathway. Surprisingly, E-selectin and IL-6 induction were no longer inhibited by MAVS knockdown when IRF-1 was also absent, revealing a redundant endothelial activation pathway. In summary, we report an IRF-1-mediated proinflammatory signaling pathway that specifically regulates LPS-mediated VCAM-1 expression, independent of NFκB., (© 2017 S. Karger AG, Basel.)

Sepsis is a systemic inflammatory response to infections associated with organ failure that is the most frequent cause of death in hospitalized patients. Exaggerated endothelial activation, altered blood flow, vascular leakage, and other disturbances synergistically contribute to sepsis-induced organ failure. The underlying signaling events associated with endothelial proinflammatory activation are not well understood, yet they likely consist of molecular pathways that act in an endothelium-specific manner. We found that LPS, a critical factor in the pathogenesis of sepsis, is internalized by endothelial cells, leading to intracellular signaling without the need for priming as found recently in immune cells. By identifying a novel role for retinoic acid-inducible gene-I (RIG-I) as a central regulator of endothelial activation functioning independent of TLR4, we provide evidence that the current paradigm of TLR4 solely being responsible for LPS-mediated endothelial responses is incomplete. RIG-I, as well as the adaptor protein mitochondrial antiviral signaling protein, regulates NF-κB-mediated induction of adhesion molecules and proinflammatory cytokine expression in response to LPS. Our findings provide essential new insights into the proinflammatory signaling pathways in endothelial cells and suggest that combined endothelial-specific inhibition of RIG-I and TLR4 will provide protection from aberrant endothelial responses associated with sepsis., (Copyright © 2016 by The American Association of Immunologists, Inc.)

Although hemodilution is attributed as the main cause of microcirculatory impairment during cardiopulmonary bypass (CPB), this relationship has never been investigated. We investigated the distinct effects of hemodilution with or without CPB on microvascular perfusion and subsequent renal tissue injury in a rat model. Male Wistar rats (375-425 g) were anesthetized, prepared for cremaster muscle intravital microscopy, and subjected to CPB (n = 9), hemodilution alone (n = 9), or a sham procedure (n = 6). Microcirculatory recordings were performed at multiple time points and analyzed for perfusion characteristics. Kidney and lung tissue were investigated for mRNA expression for genes regulating inflammation and endothelial adhesion molecule expression. Renal injury was assessed with immunohistochemistry. Hematocrit levels dropped to 0.24 ± 0.03 l/l and 0.22 ± 0.02 l/l after onset of hemodilution with or without CPB. Microcirculatory perfusion remained unaltered in sham rats. Hemodilution alone induced a 13% decrease in perfused capillaries, after which recovery was observed. Onset of CPB reduced the perfused capillaries by 40% (9.2 ± 0.9 to 5.5 ± 1.5 perfused capillaries per microscope field; P < 0.001), and this reduction persisted throughout the experiment. Endothelial and inflammatory activation and renal histological injury were increased after CPB compared with hemodilution or sham procedure. Hemodilution leads to minor and transient disturbances in microcirculatory perfusion, which cannot fully explain impaired microcirculation following cardiopulmonary bypass. CPB led to increased renal injury and endothelial adhesion molecule expression in the kidney and lung compared with hemodilution. Our findings suggest that microcirculatory impairment during CPB may play a role in the development of kidney injury., (Copyright © 2016 the American Physiological Society.)

Objective: To investigate the consequences of histone deacetylase inhibition by histone deacetylase inhibitor valproic acid and IκB kinase/nuclear factor-κB signaling blockade by IκB kinase inhibitor BAY11-7082 on (microvascular) endothelial cell behavior in vitro as well as in mice subjected to hemorrhagic shock/resuscitation in vivo., Design: Prospective, randomized laboratory investigation using an established mouse model of hemorrhagic shock., Setting: Research laboratory at university teaching hospital., Subjects: Endothelial cells and C57BL/6 male mice., Interventions: Endothelial cells were incubated with tumor necrosis factor-α in the absence or presence of valproic acid or BAY11-7082 in vitro. Mice were subjected to hemorrhagic shock by blood withdrawn until the mean arterial pressure of 30 mm Hg and maintained at this pressure for 90 minutes. At 90 minutes, subgroups of mice were resuscitated with 4% human albumin in the absence or presence of vehicle, valproic acid (300 μg/g body weight) or BAY11-7082 (400 μg per mouse). Mice were killed 1 hour and 4 hours after resuscitation., Measurements and Main Results: Valproic acid and BAY11-7082 selectively diminished tumor necrosis factor-α-induced endothelial proinflammatory activation in vitro. In vivo, both systemic and local inflammatory responses were significantly induced by hemorrhagic shock/resuscitation. The decreased histone acetylation in kidneys after hemorrhagic shock/resuscitation was restored by valproic acid treatment. In glomerular endothelial cells, the nuclear translocation of nuclear factor-κB, which was induced by hemorrhagic shock/resuscitation, was eliminated by BAY11-7082 treatment while enhanced in the presence of valproic acid. Both valproic acid and BAY11-7082 significantly attenuated the hemorrhagic shock/resuscitation-induced protein expression of endothelial cell adhesion molecules E-selectin and vascular cell adhesion molecule-1 in the microvasculature of kidneys and liver, although messenger RNA expression levels of these molecules analyzed in whole-organ lysates of kidneys, lungs, and liver were not extensively affected. The reduced protein expression of adhesion molecules was paralleled by diminishing the adhesion/transmigration of neutrophils in kidneys and liver after hemorrhagic shock/resuscitation., Conclusion: Suppression of histone deacetylase activity and blockade of IκB kinase/nuclear factor-κB signaling during resuscitation ameliorate microvascular endothelial proinflammatory responses in organs in mice after hemorrhagic shock.

Introduction: The pathophysiology of acute kidney injury (AKI) after cardiac surgery is not completely understood. Recent evidence suggests a pivotal role for the endothelium in AKI. In experimental models of AKI, the endothelial specific receptor Tie2 with its ligands Angiopoietin (Ang) 1 and Ang2 are deranged. This study investigates their status after cardiac surgery, and a possible relation between angiopoietins and AKI., Methods: From a cohort of 541 patients that underwent cardiac surgery, blood and urine was collected at 5 predefined time points. From this cohort we identified 21 patients who had at least 50% post-operative serum creatinine increase (AKI). We constructed a control group (n = 21) using propensity matching. Systemic levels of Ang1, Ang2, and sTie2 were measured in plasma and the AKI markers albumin, kidney injury molecule-1 (KIM-1) and N-acetyl-beta-D-glucosaminidase (NAG) were measured in the urine., Results: Ang2 plasma levels increased over time in AKI (from 4.2 to 11.6 ng/ml) and control patients (from 3.0 to 6.7 ng/ml). Ang2 levels increased 1.7-fold more in patients who developed AKI after cardiac surgery compared to matched control patients. Plasma levels of sTie2 decreased 1.6-fold and Ang1 decreased 3-fold over time in both groups, but were not different between AKI and controls (Ang1 P = 0.583 and sTie2 P = 0.679). Moreover, we found a positive correlation between plasma levels of Ang2 and urinary levels of NAG., Conclusions: The endothelial Ang/Tie2 system is in dysbalance in patients that develop AKI after cardiac surgery compared to matched control patients.

Coronary artery bypass graft (CABG) surgery can result in severe postoperative organ failure. During CABG surgery, cardiopulmonary bypass (CPB) with cardiac arrest is often used (on-pump CABG), which often results in a systemic inflammatory response. To reduce this inflammatory response, off-pump CABG was reintroduced, thereby avoiding CPB. There is increasing evidence that the endothelium plays an important role in the pathophysiology of organ failure after CABG surgery. In this study, 60 patients who were scheduled for elective CABG surgery were randomized to have surgery for on-pump or off-pump CABG. Blood was collected at four time points: start, end, 6 h, and 24 h postoperatively. Levels of inflammatory cytokines, soluble adhesion molecules, and angiogenic factors and their receptors were measured in the plasma. No differences were found in preoperative characteristics between the patient groups. The levels of tumor necrosis factor-α, interleukin 10, and myeloperoxidase, but not interleukin 6, were increased to a greater extent in the on-pump CABG compared with off-pump CABG after sternum closure. The soluble endothelial adhesion molecules E-selectin, vascular cell adhesion molecule 1, and intracellular adhesion molecule 1 were not elevated in the plasma during and after CABG surgery in both on-pump and off-pump CABG. Angiopoietin 2 was only increased 24 h after surgery in both on-pump and off-pump CABG. Higher levels of sFlt-1 were found after sternum closure in off-pump CABG compared with on-pump CABG. Avoiding CPB and aortic cross clamping in CABG surgery reduces the systemic inflammatory response. On-pump CABG does not lead to an increased release of soluble endothelial adhesion molecules in the circulation compared with off-pump CABG.

Rationale: Tie2 is predominantly expressed by endothelial cells and is involved in vascular integrity control during sepsis. Changes in Tie2 expression during sepsis development may contribute to microvascular dysfunction. Understanding the kinetics and molecular basis of these changes may assist in the development of therapeutic intervention to counteract microvascular dysfunction., Objective: To investigate the molecular mechanisms underlying the changes in Tie2 expression upon lipopolysaccharide (LPS) challenge., Methods and Results: Studies were performed in LPS and pro-inflammatory cytokine challenged mice as well as in mice subjected to hemorrhagic shock, primary endothelial cells were used for in vitro experiments in static and flow conditions. Eight hours after LPS challenge, Tie2 mRNA loss was observed in all major organs, while loss of Tie2 protein was predominantly observed in lungs and kidneys, in the capillaries. A similar loss could be induced by secondary cytokines TNF-α and IL-1β. Ang2 protein administration did not affect Tie2 protein expression nor was Tie2 protein rescued in LPS-challenged Ang2-deficient mice, excluding a major role for Ang2 in Tie2 down regulation. In vitro, endothelial loss of Tie2 was observed upon lowering of shear stress, not upon LPS and TNF-α stimulation, suggesting that inflammation related haemodynamic changes play a major role in loss of Tie2 in vivo, as also hemorrhagic shock induced Tie2 mRNA loss. In vitro, this loss was partially counteracted by pre-incubation with a pharmacologically NF-кB inhibitor (BAY11-7082), an effect further substantiated in vivo by pre-treatment of mice with the NF-кB inhibitor prior to the inflammatory challenge., Conclusions: Microvascular bed specific loss of Tie2 mRNA and protein in vivo upon LPS, TNFα, IL-1β challenge, as well as in response to hemorrhagic shock, is likely an indirect effect caused by a change in endothelial shear stress. This loss of Tie2 mRNA, but not Tie2 protein, induced by TNFα exposure was shown to be controlled by NF-кB signaling. Drugs aiming at restoring vascular integrity in sepsis could focus on preventing the Tie2 loss.

Background: In sepsis and various other inflammatory conditions, elevated circulating levels of angiopoietin-2 (Ang2) are detected, but the precise functional role of Ang2 in these conditions is not well understood. Here, we investigated the contribution of Ang2 to the inflammatory response and renal function impairment in a mouse model of endotoxaemia., Methods: Ang2-deficient mice and wild-type littermates were challenged with lipopolysaccharide [LPS; 1500 EU/g, intraperitoneal (i.p.)]. In additional experiments, wild-type C57Bl/6 mice were depleted of circulating neutrophils by antibody treatment (NIMPR14) prior to LPS challenge to study the role of neutrophils in regulating LPS-induced cytokine release. After 8 or 24 h of LPS challenge, the mice were sacrificed and organs were harvested. Quantitative reverse transcription polymerase chain reaction and enzyme-linked immunosorbent assay were performed for endothelial adhesion molecules (P-selectin, E-selectin, VCAM-1 and ICAM-1) and plasma cytokines (TNF-α, IL-6, KC, MIP-2), respectively. To assess renal function, blood urea nitrogen levels in plasma and albumin-to-creatinine ratio in urine were measured. RESULTS Upon LPS challenge, expression levels of various endothelial adhesion molecules in Ang2-deficient mice were reduced in an organ-specific manner. In contrast, in these mice, plasma levels of TNF-α and IL-6 were significantly increased compared with their wild-type littermates, possibly due to decreased neutrophil glomerular influx. Importantly, the absence of Ang2 did not protect the mice from acute kidney injury (AKI) upon LPS challenge. CONCLUSIONS The absence of Ang2 release upon LPS challenge induces pleotropic effects with regard to endothelial activation and systemic inflammation, but does not protect mice from LPS-induced AKI.

Introduction: The interaction between neutrophils and activated endothelium is essential for the development of multiple organ dysfunction in patients with hemorrhagic shock (HS). Mechanical ventilation frequently is used in patients with HS. The authors sought to investigate the consequences of mechanical ventilation of mice subjected to HS on microvascular endothelial activation in the lung and kidney., Methods: Anesthetized wild type C57BL/6 male mice were subjected to controlled hemorrhage; subgroups of mice were mechanically ventilated during the HS insult. To study the effect of acute hypoxia on the mice, the animals were housed in hypoxic cages. Gene expression levels was assessed by quantitative real-time polymerase chain reaction. Protein expression was assessed by immunohistochemistry and enzyme-linked immunosorbent assay., Results: Ninety minutes after the shock induction, a vascular bed-specific, heterogeneous proinflammatory endothelial activation represented by E-selectin, vascular cell adhesion molecule 1, and intercellular adhesion molecule 1 expression was seen in kidney and lung. No differences in adhesion molecules between the spontaneously breathing and mechanically ventilated mice were found. Concentrations of the proinflammatory cytokines chemokine (C-X-C motif) ligand 1 (11.0-fold) and interleukin-6 (21.7-fold) were increased after 90 min of HS. Two hours of 6% oxygen did not induce the expression of E-selectin, vascular cell adhesion molecule 1, and intercellular adhesion molecule 1 in the kidneys and the lung., Conclusions: Hemorrhagic shock leads to an early and reversible proinflammatory endothelial activation in kidney and lung. HS-induced endothelial activation is not changed by mechanical ventilation during the shock phase. Hypoxia alone does not lead to endothelial activation. The observed proinflammatory endothelial activation is mostly ischemia- or reperfusion-dependent and not related to hypoxia.

Background: As circulating plasma ATP concentrations are increased in pre-eclampsia, we tested whether increased plasma ATP is able to induce albuminuria during pregnancy., Methods: Pregnant (day 14) and non-pregnant rats were infused with ATP (3000 microg/kg bw) via a permanent jugular vein cannula. Albuminuria was determined, and blood samples were taken for leukocyte counts, plasma ATP and plasma haemopexin activity. At Day 20 of pregnancy, rats were sacrificed, fetuses and placentas weighed and kidney and placental tissue were snap frozen for immunohistology., Results: ATP infusion induced albuminuria exclusively in pregnant rats, together with increased neutrophil counts, decreased staining for glomerular sialoglycoproteins and CD39 expression, significant intraglomerular monocyte infiltration and increased glomerular intracellular adhesion molecule-1 (ICAM-1) expression. Plasma haemopexin activity was increased in saline-infused pregnant rats as compared to non-pregnant rats but was inhibited in pregnant ATP-infused rats (to non-pregnant levels). At the end of pregnancy (Day 20), increased plasma ATP level was exclusively seen in ATP-infused pregnant rats. In pregnant rats as compared with non-pregnant rats, we found decreased expression of glomerular AT-1 receptors, which was increased after ATP infusion exclusively in pregnant animals., Conclusion: The present study shows that ATP infusion induced a pro-inflammatory response leading to glomerular albuminuria exclusively in the pregnant rat. Why extracellular ATP showed this pro-inflammatory response exclusively in the pregnant condition is unclear but is probably related with relatively enhanced non-specific immunity and inflammatory reactions characteristic for the pregnant condition.

Both hemorrhagic shock and endotoxemia induce a pronounced vascular activation in the kidney which coincides with albuminuria and glomerular barrier dysfunction. We hypothesized that changes in Tie2, a vascular restricted receptor tyrosine kinase shown to control microvascular integrity and endothelial inflammation, underlie this loss of glomerular barrier function. In healthy murine and human kidney, Tie2 is heterogeneously expressed in all microvascular beds, although to different extents. In mice subjected to hemorrhagic and septic shock, Tie2 mRNA and protein were rapidly, and temporarily, lost from the renal microvasculature, and normalized within 24 h after initiation of the shock insult. The loss of Tie2 protein could not be attributed to shedding as both in mice and healthy volunteers subjected to endotoxemia, sTie2 levels in the systemic circulation did not change. In an attempt to identify the molecular control of Tie2, we activated glomerular endothelial cell cultures and human kidney slices in vitro with LPS or TNF-alpha, but did not observe a change in Tie2 mRNA levels. In parallel to the loss of Tie2 in vivo, an overt influx of neutrophils in the glomerular compartment, which coincided with proteinuria, was seen. As neutrophil-endothelial cell interactions may play a role in endothelial adaptation to shock, and these effects cannot be mimicked in vitro, we depleted neutrophils before shock induction. While this neutrophil depletion abolished proteinuria, Tie2 was not rescued, implying that Tie2 may not be a major factor controlling maintenance of the glomerular filtration barrier in this model.

During normal pregnancy, in contrast to preeclampsia, plasma hemopexin activity is increased together with a decreased vascular angiotensin II receptor (AT(1)) expression. We now tested the hypothesis that hemopexin can downregulate the AT(1) receptor in vitro. Analysis of human monocytes or endothelial cells by flow cytometry showed decreased membrane density of AT(1) exclusively after incubation with active hemopexin, whereas in supernatants of these cell cultures, AT(1) molecules could be detected (dot blotting). Also, diminished AT(1) was observed in endothelial cell lysates after contact with hemopexin (Western blotting). Hemopexin also induced extracellular signal-regulated kinase 1/2 pathway inhibition in cells after stimulation with angiotensin II in vitro, indicating downregulation of AT(1) by hemopexin. In addition, functional loss of AT(1) occurred after incubation of rat aortic rings with active hemopexin, as reflected by decreased contraction of the aortic rings on stimulation with angiotensin II. It was further demonstrated that plasma from normal pregnant women decreased the AT(1) receptor expression on monocytes as compared with plasma from nonpregnant women or preeclamptic women. Finally, it was shown that plasma hemopexin activity increases during normal gestation from week 10 onward. We concluded that active hemopexin is able to downregulate the AT(1) receptor in human monocytes, endothelial cells, and rat aortic rings. We propose that the physiological role of enhanced hemopexin activity during healthy pregnancy is to downregulate the vascular AT(1) receptor, promoting an expanded vascular bed. Inhibition of hemopexin activity during preeclampsia may result in an enhanced AT(1) receptor expression and a contracted vascular bed.

Objective: Plasma hemopexin activity, associated with increased vascular permeability, was evaluated in healthy pregnant and non-pregnant women and in pre-eclamptic women., Methods: Hemopexin activity and the hemopexin inhibitor, extracellular ATP, were assayed in plasma from pregnant (n = 10), preeclamptic (n = 9), and non-pregnant women (n = 10) using standard methods. Abdominal fascia tissue fragments from preeclamptic and pregnant women were immunohistochemically stained for vascular ecto-apyrase or ecto-5'nucleotidase., Results: The data show significantly enhanced Hx activity exclusively in plasma from pregnant women and significantly enhanced plasma ATP in pre-eclamptic women compared with the other groups. Dephosphorylation of preeclamptic plasma resulted in reactivation of Hx activity. Fascia tissue-samples from preeclamptic women showed reduced ecto-apyrase activity and enhanced ecto-5'nucleotidase activity compared to pregnant women., Conclusion: Enhanced hemopexin activity may be associated with normal pregnancy, but not with preeclampsia. Decreased hemopexin in pre-eclamptic patients may be due to enhanced plasma ATP, which is possibly promoted by diminished activity of vascular ecto-apyrase.