Plasma clot formation and clot lysis to compare effects of different anticoagulation treatments on hemostasis in patients with atrial fibrillation.

The effect of direct oral anticoagulants (DOACs) on turbidimetric measurements of plasma clot formation and susceptibility to fibrinolysis may facilitate a comparison between different classes of anticoagulants in plasma samples. We obtained 424 citrate plasma samples from 226 atrial fibrillation patients on anticoagulation and 24 samples without anticoagulation serving as controls. As comparators, we measured the international normalized ratio (INR) for phenprocoumon samples (N = 166), anti-Xa for low molecular weight heparin (LMWH) samples (N = 42), and DOAC levels with mass spectrometry (dabigatran N = 40, rivaroxaban N = 110, apixaban N = 42). Plasma clot formation and lysis were recorded continuously on a photometer after addition of an activation mix (tissue factor 2 pmol/l and tissue plasminogen activator 333 ng/ml). We used linear regression and ANCOVA for correlation analysis. Clot formation lag phase was prolonged in the presence of anticoagulants in a concentration-dependent manner for DOACs (dabigatran Spearman r = 0.74; rivaroxaban r = 0.78; apixaban r = 0.72, all p < 0.0001), INR dependent for phenprocoumon (r = 0.59, p < 0.0001), anti-Xa level dependent in LMWH samples (r = 0.90, p < 0.0001). Maximum rate of clot formation and peak clot turbidity were reduced in the presence of anticoagulants, but correlated only moderately with the comparator measures of anticoagulation. The clot lysis time was inversely correlated with DOAC concentrations in the presence of recombinant thrombomodulin. A direct ex vivo comparison between the effects of different classes of anticoagulants is possible with turbidimetric measurement of plasma clot formation and lysis. Anticoagulation inhibited clot formation in a plasma concentration manner for DOACs, INR dependent for phenprocoumon, and anti-Xa dependent for LMWH. Susceptibility to fibrinolysis increased with increasing DOAC concentrations.

Underlying mechanism and specific prevention of hemolysis-induced platelet activation.

Thromboembolic complications significantly impair the outcome of hemolytic disorders. We hypothesized that red cell adenosine diphosphate (ADP) release results in significant platelet activation in hemolysis and that this prothrombotic state can be prevented by inhibition of the ADP P2Y12 receptor. In the current study, we therefore sought to investigate the mechanism and inhibition of hemolysis-induced platelet activation. The expression of activated integrin αIIbß3 was determined by flow cytometry, and platelet aggregation was assessed by multiple electrode platelet aggregometry. We demonstrate platelet activation and increased platelet aggregation by adding hemolytic blood (lysates) to whole blood, similarly to that achieved by the platelet agonist ADP. Enhanced platelet activation and reactivity in the presence of hemolytic blood were significantly abolished by apyrase, which catalyzes ADP degradation, and inhibited by blockade of the platelet ADP P2Y12 receptor with cangrelor. Platelets from patients treated with the ADP P2Y12 receptor antagonist clopidogrel showed a reduced response to lysates compared to platelets from healthy controls without antiplatelet treatment. Further, in vitro blood group ABO incompatibility induced hemolysis and led to increased platelet activation. Finally, "spontaneous" platelet aggregation seen in patients with cold agglutinin disease was completely abolished by cangrelor. In conclusion, hemolysis is associated with increased platelet activation and aggregation due to red cell derived ADP, which can be prevented by ADP receptor blockade.

Human immune-cell response towards diverse xenogeneic and allogeneic decellularized biomaterials.

BACKGROUND: Immunological knowledge on processed biological implants and mesh-prostheses is still mainly based on animal models, lacking information on the species-specific human immune response. We hypothesized that in contrast to human tissue even decellularized xenogenic specimens would lead to significant and tissue source dependent human immune reactions. METHODS: Specimens from processed allogenic and xenogenic pulmonary arteries, pericardium or dermis, were co-cultured with human peripheral blood mononuclear cells (PBMNC). Proliferative responses were measured in tritiated thymidine incorporation assays (n = 10). Stimulation indices (SI), calculated as counts-per-minute of co-cultured PBMNC divided by the cpm of basic cell proliferation, were compared. RESULTS: Compared to native porcine pulmonary artery tissue decellularization significantly reduced human PBMNC proliferation (mean SI: 48.7 vs. 18.0, p < 0.01), which was still higher compared to the human equivalent (SI: 0.7 vs. 1.7). Also the processed human dermal implant did not elicit immune response (SI: 1.5), whereas the decellularized and cross-linked porcine dermis lead to a significant human cell-proliferation (SI: 8.4, p < 0.01). Interestingly, both the processed human (SI: 15.2) and bovine pericardial patches (SI: 15.1) led to higher immune cell proliferation. CONCLUSION: Even decellularized or cross-linked xenogenic cardiovascular and reconstructive biomaterials elicit increased human immune responses not seen in the majority of allogenic specimens tested.

Anti-coagulation assessment with prothrombin time and anti-Xa assays in real-world patients on treatment with rivaroxaban.

Monitoring of anti-coagulation with the direct factor Xa inhibitor rivaroxaban is considered unnecessary in a routine clinical setting. However, assessment of its anti-coagulant effect may be desirable in certain clinical situations. We assessed prothrombin time (PT) reagents and commercially available anti-Xa assays (Biophen) calibrated for rivaroxaban and heparin in comparison to liquid chromatography-mass spectrometry (LC-MS/MS) measurements of rivaroxaban concentration in samples from patients on treatment with rivaroxaban for stroke prevention in atrial fibrillation. Citrate plasma samples were obtained from 30 randomly selected patients on uninterrupted treatment with rivaroxaban for a minimum of 1 month. The anti-Xa assays, direct Xa inhibitor (DiXa-I®), and Heparin LRT® were conducted for both wide and low calibrations for rivaroxaban. Measurements were compared to LC-MS/MS using correlation, linear regression, intra-class correlation, and Bland-Altman analysis. In 30 patients (9 female) of median age 71.5 years and BMI 26.5 kg/m(2), rivaroxaban concentrations between 2.4 and 625 ng/ml (median 82 ng/ml) were measured by LC-MS/MS. PT reagents were poorly correlated with rivaroxaban concentrations (r (2) = 0.52 and 0.09). Anti-Xa assays DiXa-I (r (2) = 0.95) and Heparin LRT (r (2) = 0.97) were correlated with rivaroxaban in all concentrations, but especially in low concentrations with low calibrations (r (2) = 0.97 and 0.98, respectively). The highest agreement occurred between Heparin LRT and low rivaroxaban concentrations with a mean difference of -5.3 ng/ml (limits of agreement, 12.9 to 2.4 ng/ml). Anti-Xa assays can indirectly determine the concentration of rivaroxaban for a wide range of concentrations in real-world patients. An interpretation of anti-Xa and PT measurements in treatment with rivaroxaban requires knowledge of the local reagents.

Identification of Aspergillus fumigatus Surface Components That Mediate Interaction of Conidia and Hyphae With Human Platelets.

BACKGROUND: Platelets were recently identified as a part of innate immunity. They are activated by contact with Aspergillus fumigatus; putative consequences include antifungal defense but also thrombosis, excessive inflammation, and thrombocytopenia. We aimed to identify those fungal surface structures that mediate interaction with platelets. METHODS: Human platelets were incubated with Aspergillus conidia and hyphae, isolated wall components, or fungal surface mutants. Interaction was visualized microscopically; activation was quantified by flow cytometry of specific markers. RESULTS: The capacity of A. fumigatus conidia to activate platelets is at least partly due to melanin, because this effect can be mimicked with "melanin ghosts"; a mutant lacking melanin showed reduced platelet stimulating potency. In contrast, conidial hydrophobin masks relevant structures, because an A. fumigatus mutant lacking the hydrophobin protein induced stronger platelet activation than wild-type conidia. A. fumigatus hyphae also contain surface structures that interact with platelets. Wall proteins, galactomannan, chitin, and ß-glucan are not the relevant hyphal components; instead, the recently identified fungal polysaccharide galactosaminogalactan potently triggered platelet activation. CONCLUSIONS: Conidial melanin and hydrophobin as well as hyphal galactosaminogalactan represent important pathogenicity factors that modulate platelet activity and thus might influence immune responses, inflammation, and thrombosis in infected patients.

Administration of recombinant human granulocyte-colony-stimulating factor does not induce long-lasting detectable epigenetic alterations in healthy donors.

BACKGROUND: The short-term safety profile of recombinant human granulocyte-colony-stimulating factor (rHuG-CSF) in the allogeneic stem cell setting seems acceptable; only few data on long-term safety are available. To further study possible epigenetic alterations, we investigated prospectively the influence of rHuG-CSF on DNA methyltransferase (DNMT) activity and on changes in DNA methylation of candidate genes in peripheral blood cells of healthy unrelated stem cell donors within an observation period of 1 year. STUDY DESIGN AND METHODS: In this study, 20 stem cell donors (14 male/six female; median age, 40 years; range, 22-54 years) and 20 sex- and age-matched blood component donors (controls) were included. Sampling was performed before rHuG-CSF administration; at the time of donation; and on Days (+1), 7, 30, 100, 180, and 360 in both groups. Analysis of DNMT activity in nuclear extracts was performed using a modified radionuclide assay. We performed methylation-specific polymerase chain reaction to detect the methylation status of promoter CpG islands of the genes of the retinoic acid receptor beta (RAR-B) and the Ras association domain family 1A (RASSF1A). RESULTS: DNMT activity increased significantly on the day of donation and 1 day after (p < 0.05). By Day +7 baseline values were reached. No further significant alterations of DNMT activity in the treated group compared to the controls were observed. We could not detect any differences in the gene methylation of RAR-B and RASSF1A between both groups. CONCLUSION: In our prospective study no evidence of long-lasting increased DNMT activity or enhanced DNA methylation in a limited panel of target genes after recombinant human G-CSF administration was observed in healthy stem cell donors.

Native multimer analysis of plasma and platelet von Willebrand factor compared to denaturing separation: implication for the interpretation of satellite bands.

Blue native electrophoresis (BNE) was applied to analyze the von Willebrand factor (vWF) multimers in their native state and to present a methodology to perform blue native electrophoresis on human plasma proteins, which has not been done before. The major difference between this method and the commonly used SDS-agarose gel electrophoresis is the lack of satellite bands in the high-resolution native gel. To further analyze this phenomenon, a second dimension was performed under denaturing conditions. Thereby, we obtained a pattern in which each protein sub-unit from the first dimension dissociates into three distinct sub-bands. These bands confirm the triplet structure, which consists of an intermediate band and two satellite bands. By introducing the second dimension, our novel method separates the triplet structure into a higher resolution than the commonly used SDS-agarose gel electrophoresis does. This helps considerably in the classification of ambiguous von Willebrand's disease subtypes. In addition, our method has the additional advantage of being able to resolve the triplet structure of platelet vWF multimers, which has not been identified previously through conventional SDS-agarose electrophoresis multimer analysis. This potential enables us to compare the triplet structure from platelet and plasmatic vWF, and may help to find out whether structural abnormalities concern the vWF molecule in the platelet itself, or whether they are due to the physiological processing of vWF shed into circulation. Owing to its resolution and sensitivity, this native separation technique offers a promising tool for the analysis and detection of von Willebrand disorder, and for the classification of von Willebrand's disease subtypes.

P-selectin mRNA is maintained in platelet concentrates stored at 4 degrees C.

BACKGROUND: Platelets (PLTs) contain mRNA and synthesize proteins in response to activation. Most guidelines for PLT concentrates (PCs) recommend ambient temperature for storage but the impact of the storage temperature on PLT mRNA content has not yet been investigated. STUDY DESIGN AND METHODS: Ten leukoreduced apheresis PCs were split and stored at 22 and 4 degrees C. P-selectin mRNA, its expression on PLTs, and its soluble form were quantified. In parallel, cellular (cell count, mean PLT volume), metabolic (pH, pO(2), pCO(2), HCO(3), glucose), and functional markers (swirling, hypotonic shock response, aggregation to collagen) were analyzed. Rotation thrombelastography was used to monitor the hemostatic potential of PLTs. All measurements were performed on Days 1 and 5 of storage. RESULTS: After 5 days of storage at 4 degrees C, only 31 +/- 27 percent of P-selectin mRNA and 29 +/- 41 percent of glyceraldehyde-3-phosphate dehydrogenase mRNA were lost, while minute amounts of the mRNAs were detectable at 22 degrees C. In PCs stored at 4 degrees C the percentage of P-selectin-positive PLTs was significantly higher when compared to PCs stored at 22 degrees C. Soluble P-selectin concentrations did not significantly differ between both storage temperatures. Thrombelastography revealed significantly shorter reaction times in PLTs kept at 4 degrees C. CONCLUSION: Our data indicate that storage at 4 degrees C is accompanied by maintained mRNA levels. PLTs with intact mRNA levels and short reaction times in thrombelastography might be functionally superior to PLTs that are devoid of mRNA and show less augmented P-selectin surface expression. In therapeutic applications, that is, if PLTs are transfused to control acute bleeding, PLTs kept at 4 degrees C may be advantageous.

Biomechanical properties of decellularized porcine pulmonary valve conduits.

Tissue-engineered heart valves constructed from a xenogeneic or allogeneic decellularized matrix might overcome the disadvantages of current heart valve substitutes. One major necessity besides effective decellularization is to preserve the biomechanical properties of the valve. Native and decellularized porcine pulmonary heart valve conduits (PPVCs) (with [n = 10] or without [n = 10] cryopreservation) were compared to cryopreserved human pulmonary valve conduits (n = 7). Samples of the conduit were measured for wall thickness and underwent tensile tests. Elongation measurement was performed with a video extensometer. Decellularized PPVC showed a higher failure force both in longitudinal (+73%; P < 0.01) and transverse (+66%; P < 0.001) direction compared to human homografts. Failure force of the tissue after cryopreservation was still higher in the porcine group (longitudinal: +106%, P < 0.01; transverse: +58%, P < 0.001). In comparison to human homografts, both decellularized and decellularized cryopreserved porcine conduits showed a higher extensibility in longitudinal (decellularized: +61%, P < 0.001; decellularized + cryopreserved: +51%, P < 0.01) and transverse (decellularized: +126%, P < 0.001; decellularized + cryopreserved: +118%, P < 0.001) direction. Again, cryopreservation did not influence the biomechanical properties of the decellularized porcine matrix.

Reactivity of integrin-linked kinase in human mesothelial cell proliferation.

Integrin-linked kinase (ILK) is a protein kinase that links integrins and growth factors to a range of signalling pathways. ILK expression and activity are increased in a variety of human cancers. However, little is known regarding the role of ILK in malignant pleural mesothelioma (MPM). In this study, we assessed the expression of ILK in samples of human MPM, and compared it with the expression of epidermal growth factor receptor (EGFR). Thirty-four samples of human malignant mesothelioma were stained with a polyclonal antibody against ILK. Two independent observers evaluated the morphological pattern and intensity of staining. The findings have been compared with the patient's characteristics. Most MPM and mesothelial cell proliferation samples (87.9%) showed cytoplasmic ILK staining of varying intensity. Normal mesothelial cells and normal lung parenchyma did not stain for ILK at all. Conversely, the percentage of positive EGFR staining was somewhat lower (75.8%). The ILK-positive patients were significantly older than the ILK-negative patients. Here we report for the first time that ILK is indeed expressed in malignant mesothelioma. For further validation of a causal association between ILK and neoplastic mesothelial transformation, these immunohistochemical results should be supplemented with clinical and molecular biological data.

Recent trends in heart transplantation: the University of Vienna experience.

Since the beginning of the University of Vienna Cardiac Transplant Program in 1984, 1086 heart transplant procedures have been performed through the end of 2007. One- and five-year survival has increased steadily over time (82% and 76%). Ten-year survival is 65%. Over the past 10 years our program has seen dramatic changes in patient selection, accepting now patients with more risk factors (Age, diabetes, elevated pulmonary resistance,..). Developments in immunosuppression have decreased incidence of infection, rejection and graft arteriosclerosis continuously. Our program continues to pursue novel strategies to improve the survival and quality of life of our heart transplant patients.

Granulocyte-based immune response against decellularized or glutaraldehyde cross-linked vascular tissue.

Supporting structures derived from biological tissue have been used in numerous tissue-engineering applications. This study focuses on the immune response of human leukocytes toward decellularized or glutaraldehyde (GA) cross-linked vascular tissue in vitro. Porcine and human pulmonary roots were sterilized with antibiotics, decellularized or cross-linked with GA. Proteins of the vascular tissue were extracted and the migratory response of human leukocytes toward protein extracts was examined using an in vitro migration chamber. Transmigrated leukocytes were counted and subsets (lymphocytes, monocytes, granulocytes) analyzed by flow cytometry. Decellularization significantly reduced the migration of monocytes compared to native porcine tissue. Although the proportion of transmigrating lymphocytes was much lower, decellularization again reduced the migratory response. Surprisingly, after decellularization granulocyte migration was still significantly higher than the negative control. Results comparable to those obtained with porcine material were found when human tissue was used for the experiments. Interestingly, migratory behavior toward extracts of GA-fixed porcine tissue was similar to that of decellularized specimens. We have shown that decellularization of vascular tissue reduces lymphocyte and monocyte recruitment comparable to cross-linking treatment. However, the migration of granulocytes, which are also known to be strongly involved in early inflammatory reactions, could be abolished neither by decellularization nor by fixation with GA.

Plectin scaffolds recruit energy-controlling AMP-activated protein kinase (AMPK) in differentiated myofibres.

Plectin, a cytolinker protein greater than 500 kDa in size, has an important role as a mechanical stabiliser of cells. It interlinks the various cytoskeletal filament systems and anchors intermediate filaments to peripheral junctional complexes. In addition, there is increasing evidence that plectin acts as a scaffolding platform that controls the spatial and temporal localisation and interaction of signaling proteins. In this study we show that, in differentiated mouse myotubes, plectin binds to the regulatory gamma1 subunit of AMP-activated protein kinase (AMPK), the key regulatory enzyme of energy homeostasis. No interaction was observed in undifferentiated myoblasts, and plectin-deficient myotubes showed altered positioning of gamma1-AMPK. In addition we found that plectin affects the subunit composition of AMPK, because isoform alpha1 of the catalytic subunit decreased in proportion to isoform alpha2 during in vitro differentiation of plectin(-/-) myotubes. In plectin-deficient myocytes we could also detect a higher level of activated (Thr172-phosphorylated) AMPK, compared with wild-type cells. Our data suggest a differentiation-dependent association of plectin with AMPK, where plectin selectively stabilises alpha1-gamma1 AMPK complexes by binding to the gamma1 regulatory subunit. The distinct plectin expression patterns in different fibre types combined with its involvement in the regulation of isoform compositions of AMPK complexes could provide a mechanism whereby cytoarchitecture influences energy homeostasis.

Depletion of endothelial progenitor cells in the peripheral blood of patients with rheumatoid arthritis.

BACKGROUND: Rheumatoid arthritis (RA) is characterized by increased cardiovascular morbidity and mortality that cannot be explained solely by traditional cardiovascular risk factors. Cardiovascular morbidity is related to disease activity and can be normalized by effective therapy. Because the quantity of endothelial progenitor cells (EPCs) in the peripheral blood is correlated inversely with cardiovascular risk, we studied whether such abnormalities could also be observed in patients with RA. METHODS AND RESULTS: EPCs were determined in 52 RA patients and in 16 healthy referents (HRs) by fluorescence-activated cell-sorting (FACS) analysis. Patients were divided into groups characterized by active disease (n=36) and low disease activity (n=16). Cells that were positive by flow cytometry for CD34/KDR/AC133 within the lymphocyte population were characterized as EPCs. Furthermore, in subgroups of patients, circulating EPCs were also quantified by a colony-forming unit (CFU) and a circulating angiogenic cell (CAC) assay. EPCs were significantly decreased in RA patients suffering from active disease compared with those from HRs, as measured by FACS analysis (0.026+/-0.002% versus 0.045+/-0.008%, respectively, P<0.05), CFU assay (mean of 5+/-2 versus 18+/-5 CFU/well in HRs, P<0.05), and CAC assay (mean of 7+/-2 versus 52+/-16 positive cells/high-power field, P<0.005). In contrast, the frequency of circulating EPCs from patients with low disease activity was comparable to that of healthy individuals (0.052+/-0.006% by FACS analysis), CFU assay (10+/-5 CFU/well), and CAC assay (mean of 25+/-5 positive cells). Moreover, EPC quantities in peripheral blood were correlated inversely with disease activity as assessed by the disease activity score (r=-0.38, P<0.01). CONCLUSIONS: Our observations indicate that active RA is associated with a depletion of circulating EPCs. This might be one of several factors contributing to the increased cardiovascular risk in RA.

Ferrous sulfate does not affect mycophenolic acid pharmacokinetics in kidney transplant patients.

BACKGROUND: Oral administration of ferrous-sulfate was reported to decrease intestinal absorption of mycophenolate mofetil (MMF) in healthy Japanese individuals by 90%. METHODS: We examined the effect of a single oral dose of ferrous sulfate on steady-state mycophenolic acid pharmacokinetics in 10 iron-deficient (hypochromic red blood cells >2.5%), Caucasian, long-term kidney graft recipients using a randomized, open-label, crossover design. On days A and B, MMF (1,000 mg) was given orally at 8:00 am. On day C, MMF and ferrous sulfate (105 mg) were coadministered at 8:00 am. On day D, MMF was given at 8:00 am and ferrous sulfate was given orally 4 hours later. RESULTS: The interindividual variability of the 12-hour area under the plasma mycophenolic acid concentration versus time curves (AUC(0-12)) under control conditions was small (89.5 +/- 27.8 and 87.6 +/- 39.1 mg x h/L, respectively). Concomitant or subsequent administration of MMF and ferrous sulfate did not affect the bioavailabilty of MMF (AUC(0-12), 91.9 +/- 30.4 mg x h/L and 96.0 +/- 31.7 mg x h/L). CONCLUSION: Oral therapy of iron deficiency using ferrous sulfate in long-term kidney graft recipients does not impede intestinal absorption of MMF; hence, exposure to this immunosuppressive agent is not reduced.

Establishment of vessel-like structures in long-term three-dimensional tissue culture of myocardium: an electron microscopy study.

To assess whether long-term three-dimensional (3D) tissue culture of myocardium enables the in vitro establishment of vessel-like structures, myocardial tissue from newborn mice was incubated under conditions of 3D culture for at least 3 weeks, and studied by phase-contrast microscopy, conventional histology, immunohistochemistry, and electron microscopy. During 3 weeks of culture, a mean 24.35 +/- 3.74% of all aggregates contracted spontaneously. The contracting aggregates displayed a tissue-like architecture with small basal and apical zones, and a large central zone. The basal and apical zone consisted of immature mesenchymal cells. The underlying shell of the aggregate contained many cardiomyocytes. Vessel-like structures were found concentrated within the aggregates. Immunohistochemistry showed that up to 15% of the cells in the central zone of the aggregate were positive for the endothelial-specific BS-I lectin. Vessel-like structures were formed by cells, which often showed intracytoplasmatic lumena. Surrounding the neocapillaries, structures of a rudimentary basal membrane could be detected. A 3D culture of myocardial tissue permits the establishment of a rudimentary capillary network within the tissue aggregates, which presumably guarantees a sufficient tissue perfusion up to a maximum aggregate diameter of approximately 900 microm.

Modulation of toll-like receptor 4 expression on human monocytes by tumor necrosis factor and interleukin-6: tumor necrosis factor evokes lipopolysaccharide hyporesponsiveness, whereas interleukin-6 enhances lipopolysaccharide activity.

Toll-like receptors (TLR) play a pivotal role in the innate immune response, and the expression levels of these receptors may reflect the sensitivity of immune cells to infections. The binding of lipopolysaccharide (LPS) to TLR-4 triggers human monocytes to produce cytokines, which play a dominant role in the inflammatory response, as can be observed during sepsis and after polytrauma. Here, we evaluated TLR-4 expression of isolated monocytes in the presence of tumor necrosis factor (TNF)-alpha, interleukin (IL) 6, IL-8, and IL-10, and we investigated cellular activation of this treatment. TNF-alpha significantly down-regulated TLR-4 mRNA expression after 6 h (100% vs. 38.5% +/- 4%; P < 0.05). This down-regulation was followed by a dose- and time-dependent diminished expression of TLR-4 surface protein (100% vs. 8.0% +/- 5%; P < 0.01). Forty-eight hours after TNF-alpha treatment, a reduced nuclear factor (NF)-kappaB translocation and a diminished IL-6 secretion after LPS stimulation were found (100% vs. 42.0% +/- 23%; P < 0.05). In contrast, IL-6 incubation upregulated TLR-4 cell surface protein (100% vs. 165.8% +/- 24%; P < 0.05) and increased the ability to activate NF-kappaB and AP-1 after LPS stimulation. Stimulation with IL-8 or IL-10 had no significant effects. We conclude that not only LPS but also TNF-alpha and IL-6 have the potency to regulate the immune response via TLR-4. Down-regulation of TLR-4 by TNF-alpha is associated with LPS hyporeactivity for NF-kappaB formation, whereas upregulation of TLR-4 via IL-6 can increase the responsiveness of mononuclear phagocytes.

Effect of prostaglandin-J(2) on VEGF synthesis depends on the induction of heme oxygenase-1.

Heme oxygenase-1 (HO-1) is an inducible enzyme that degrades heme to carbon monoxide, iron ions, and biliverdin. Its expression can be induced by 15-deoxy-Delta(12,14)prostaglandin-J(2) (15d-PGJ(2)), a natural ligand of peroxisome proliferator-activated receptor-gamma transcription factor. In macrophages and vascular smooth muscle cells, 15d-PGJ(2) up-regulates the expression of vascular endothelial growth factor (VEGF), a fundamental regulator of angiogenesis. Here we investigated the involvement of HO-1 in the 15d-PGJ(2)-mediated regulation of VEGF production by human microvascular endothelial cells (HMEC-1). Resting HMEC-1 released approximately 20 pg/ml VEGF protein after 24 h of incubation. Treatment of cells with 15d-PGJ(2) (1-10 microM) significantly and dose-dependently increased the VEGF promoter activity, mRNA expression, and protein secretion. In the same cells, 15d-PGJ(2) potently induced the expression of HO-1 protein that correlated with HO-1 promoter activity. Activation of HO-1 with hemin or ectopic overexpression of HO-1 in HMEC-1 perfectly mimicked the effect of 15d-PGJ(2) and led to increased VEGF production. Importantly, the inhibition of the HO-1 pathway by tin protoporphyrin-IX significantly reduced the stimulatory effect of 15d-PGJ(2) on VEGF synthesis. Thus, we postulate that the up-regulation of VEGF expression in response to 15d-PGJ(2 )in HMEC-1 is mediated by the activation of HO-1.