Tobias Hilbert

Priming with Synthetic Oligonucleotides Attenuates Pressure Overload-Induced Inflammation and Cardiac Hypertrophy in Mice

AIMS Inflammation and Toll-like receptor (TLR) signalling have been linked to the development of cardiac hypertrophy following transverse aortic constriction (TAC). In the present study, we investigated whether pre-treatment with the synthetic TLR9 ligands 1668-thioate or 1612-thioate modulates the progression of TAC-induced cardiac inflammation and hypertrophy. METHODS AND RESULTS C57BL/6N-mice were pre-treated with 1668-thioate, 1612-thioate (0.25 nmol/g, i.p.), or phosphate-buffered saline 16 h prior to TAC or sham surgery. Heart-weight/body-weight ratio (HW/BW), cardiomyocyte cell size, cellular macrophage accumulation, myofibroblast differentiation, and collagen deposition were investigated for up to 28 days. Cardiac function was monitored using a pressure-volume catheter and M-mode echocardiography. Inflammatory gene expression in the heart was analysed via gene array, while the time course of mRNA expression of key inflammatory mediators was assessed via RT-qPCR. TAC increased the HW/BW ratio and cardiomyocyte cell size and induced macrophage accumulation, myofibroblast differentiation, and collagen deposition. These changes were accompanied by cardiac inflammation and a significant loss of left ventricular function. Pre-treatment with cytosine-phosphate-guanine (CpG)-containing 1668-thioate attenuated the inflammatory response, the progression of cardiac hypertrophy, and cardiac remodelling, which resulted in a prolonged preservation of left ventricular function. These changes were induced to a smaller extent by the use of the non-CG-containing oligodeoxynucleotide 1612-thioate. CONCLUSION Pre-treatment with 1668-thioate attenuated cardiac hypertrophy following pressure overload, possibly by modifying the hypertrophy-induced inflammatory response, thereby reducing cardiac growth and fibrosis as well as delaying loss of cardiac function.

Effective angiostatic treatment in a murine metastatic and orthotopic hepatoma model

Vascular endothelial growth factor (VEGF) activity is correlated with a progressive tumor disease in patients with hepatocellular carcinoma (HCC). In spite of the well‐recognized role of VEGF in HCC, there are few data available regarding therapeutic strategies to block VEGF activity. Therefore, we employed a recombinant adenoviral vector encoding a soluble dominant negative fragment of VEGF receptor 2 (Flk‐1), AdsFlk‐1, to control pre‐established murine orthotopic and metastatic hepatomas. Vector function was confirmed via reverse‐transcriptase polymerase chain reaction and ELISA, and angiostatic effects were analyzed in vitro and in vivo. Antitumoral effects of systemic AdsFlk‐1 application were studied in a subcutaneous and orthotopic Hepa129 HCC model. Cell supernatant containing the truncated form of Flk‐1 had no direct effect on cell proliferation of Hepa129 cells in vitro but reduced endothelial tube formation on matrigel matrix by approximately 80% in vitro. Endothelial‐like cell infiltration into matrigel plugs in vivo was also decreased by 80%. Systemic treatment of tumor‐bearing mice inhibited tumor growth by 84% compared with the corresponding control group within 16 days after vector application. Likewise, the survival rate was significantly improved in the AdsFlk‐1 group compared with control. Orthotopic tumor growth was reduced by 82%, and development of malignant ascites was also retarded. In conclusion, systemic adenoviral‐mediated gene transfer of an Flk‐1 fragment significantly inhibited tumor growth in orthotopic and metastatic murine HCC. The data support the value of VEGF blockade as an effective target for HCC treatment. (HEPATOLOGY 2005;41:1233–1240)

Anti-atherogenic effects of statins: Impact on angiopoietin-2 release from endothelial cells

Beyond lipid lowering, statins are supposed to exert pleiotropic effects positively influencing the progression of atherosclerotic lesions. The development of such lesions is associated with increased release of angiopoietin-2 (Ang-2), an endothelial cell-specific protein growth factor stored in Weibel-Palade bodies (WPBs). The aim of our study was to examine whether statin pretreatment influences the release of Ang-2 from endothelial cells. Stimulation of HUVECs and HMVECs with PMA, thrombin or histamine resulted in significant release of Ang-2, as evidenced by ELISA. Pretreatment with simvastatin and mevastatin suppressed this release to basal level, while pravastatin had no effect. Simvastatin itself increased nitric oxide (NO, EC number 1.14.13.39) synthesis, measured by Griess reaction. Combining the statin pretreatment with the eNOS inhibitor L-NNA as well as bypassing the HMG-CoA reductase (EC number: 1.1.1.34) by adding mevalonic acid or geranyl pyrophosphate restored the exocytotic effect of PMA. Immunofluorescence microscopy showed that depletion of WPBs upon PMA stimulation ceased after pretreatment with simvastatin. This study demonstrates a potent suppressive effect of statins on the release of Ang-2 from endothelial cells. Regarding its harmful effects in the development of atherosclerotic lesions, our data provide further insight into the mechanisms of the anti-atherogenic potential of statins.

The angiopoietin/TIE receptor system: Focusing its role for ischemia-reperfusion injury

Ischemia and reperfusion (I/R) are of fatal consequence for the affected organs, as they provoke a profound inflammatory reaction. This thoroughly destroys cells and tissues, inducing functional failure or even complete loss of organ function. Since I/R is primarily a vascular problem, the interaction between the endothelium and the surrounding environment is of great significance. The angiopoietins (ANG) and the TIE receptors are key players for the vascular homeostasis. This review summarizes biochemical and cellular mechanisms leading to I/R injury. After a brief introduction to the ANG/TIE system, a comprehensive overview of its role for the development of I/R syndrome is given. Finally, current therapeutic approaches to mitigate the consequences of I/R by modulating ANG/TIE signaling are reviewed in detail.

Beta2-adrenoceptor stimulation suppresses TLR9-dependent IFNA1 secretion in human peripheral blood mononuclear cells.

Introduction IFNA1 (interferon alpha) is a key cytokine regulating the activity of numerous immune cells. Plasmacytoid dendritic cells (pDCs) as natural interferon-producing cells play critical roles as sensors of pathogens and link innate to adaptive immunity. CpG motifs within DNA sequences activating toll-like receptor 9 (TLR9) are the main stimuli eliciting IFNA1 secretion from pDCs. Adrenergic substances are capable of differentially modulating the response from various immune cells. Hence, the aim of this study was to examine how adrenoceptor stimulation influences TLR9-induced IFNA1 secretion from human pDCs. Methods PBMCs generated from human whole blood and pDCs enriched from buffy coats were stimulated with LPS and CpG-ODN 2336 in the presence or absence of epinephrine and different adrenoceptor antagonists. Secretion of TNF and IFNA1 was measured by ELISA. Flow cytometry was used to determine efficacy of pDC enrichment and adrenoceptor expression of PBMC subsets. The influence of modified IFNA1 secretion on NK cell activity was evaluated using a colorimetric tumor cell lysis assay. Results TLR9-induced IFNA1 secretion as well as TLR4-induced TNF secretion from PBMCs was dose-dependently attenuated by coincubation with epinephrine. Combination with different specific adrenoceptor antagonists revealed that this effect was mediated by the adrenoceptor β2 (ADRB2). Since flow cytometric analysis could exclude the presence of ADRB2 on pDCs, highly enriched pDCs lacked any visible impact of adrenoceptor stimulation on TLR9-induced IFNA1 release. Combination of pDCs with PBMCs restored the effect, even when they were separated by a permeable membrane. Suppression of TLR9-mediated IFNA1 secretion from PBMCs by adrenoceptor stimulation reduced the lytic activity of NK cells on K562 tumor cells. Conclusion We provide insights into the underlying mechanisms of the interrelation between immune responses and pharmacological agents widely used in clinical practice. Our results have implications for the future treatment of human patients, in which the endogenous immune response plays a pivotal role, such as during viral infections, inflammatory diseases and cancers.

Pulmonary vascular inflammation: effect of TLR signalling on angiopoietin/TIE regulation.

Increased pulmonary vascular resistance is a critical complication in sepsis. Toll‐like receptor (TLR) as well as angiopoietin (ANG) signalling both contribute to the emergence of pulmonary arterial hypertension. We hypothesized that TLR stimulation by bacterial ligands directly affects expression and secretion of ligands and receptors of the angiopoietin/TIE axis. Microvascular endothelial (HPMEC) and smooth muscle cells (SMC) of pulmonary origin were incubated with thrombin and with ligands for TLR2, ‐4, ‐5, and ‐9. Expression and secretion of ANG1, ‐2, TIE2 and IL‐8 were determined using quantitative real‐time PCR and ELISA. TLR stimulation had no impact either on the expression of ANG2 and TIE2 in HPMEC or on that of ANG1 in SMC. However, overall levels of both released ANG1 and ‐2 were halved upon stimulation with the TLR9 ligand CpG, and ANG2 release was significantly enhanced by TLR4 activation when initially provoked by sequentially performed stimulation. Furthermore, enhanced ANG2 activity increased endothelial permeability, as demonstrated in an in vitro transwell assay. We conclude that sole TLR stimulation by bacterial ligands plays no significant role for altered expression and secretion of ANG1, ‐2 and TIE2 in human pulmonary vascular cells. The interplay between various stimuli is required to induce imbalances between ANG1 and ‐2.

Is internal jugular vein extensibility associated with indices of fluid responsiveness in ventilated patients

Ultrasound of the inferior vena cava provides rapid and non‐invasive assessment of fluid responsiveness. We hypothesized that the extensibility of the internal jugular vein (IJV) as well reflects intravascular volume state. We assessed IJV dimensions together with pulse pressure variation (PPV) as dynamic index for fluid responsiveness in mechanically ventilated patients.

Synthetic CpG oligonucleotides induce a genetic profile ameliorating murine myocardial I/R injury

We previously demonstrated that pre‐conditioning with CpG oligonucleotide (ODN) 1668 induces quick up‐regulation of gene expression 3 hours post‐murine myocardial ischaemia/reperfusion (I/R) injury, terminating inflammatory processes that sustain I/R injury. Now, performing comprehensive microarray and biocomputational analyses, we sought to further enlighten the “black box” beyond these first 3 hours. C57BL/6 mice were pretreated with either CpG 1668 or with control ODN 1612, respectively. Sixteen hours later, myocardial ischaemia was induced for 1 hour in a closed‐chest model, followed by reperfusion for 24 hours. RNA was extracted from hearts, and labelled cDNA was hybridized to gene microarrays. Data analysis was performed with BRB ArrayTools and Ingenuity Pathway Analysis. Functional groups mediating restoration of cellular integrity were among the top up‐regulated categories. Genes known to influence cardiomyocyte survival were strongly induced 24 hours post‐I/R. In contrast, proinflammatory pathways were down‐regulated. Interleukin‐10, an upstream regulator, suppressed specifically selected proinflammatory target genes at 24 hours compared to 3 hours post‐I/R. The IL1 complex is supposed to be one regulator of a network increasing cardiovascular angiogenesis. The up‐regulation of numerous protective pathways and the suppression of proinflammatory activity are supposed to be the genetic correlate of the cardioprotective effects of CpG 1668 pre‐conditioning.

Experimental murine acute lung injury induces increase of pulmonary TIE2-expressing macrophages

BackgroundBreakdown of the alveolo-capillary wall is pathognomonic for Acute Lung Injury (ALI). Angiopoietins, vascular-specific growth factors, are linked to endothelial barrier dysfunction, and elevated Angiopoietin-2 (ANG2) levels are associated with poor outcome of ALI patients. Specialized immune cells, referred to as ‘TIE2-expressing monocytes and macrophages’ (TEM), were shown to specifically respond to ANG2 binding. However, their involvement in acute inflammatory processes is so far completely undescribed. Thus, our aim was to assess the dynamics of TEMs in a murine model of ALI.ResultsIntratracheal instillation of LPS induced a robust pulmonary pro-inflammatory response with endothelial barrier dysfunction and significantly enhanced ANG2 expression. The percentage number of TEMs, assessed by FACS analysis, was more than trebled compared to controls, with TEM count in lungs reaching more than 40% of all macrophages. Such distinct dynamic was absent in all other analyzed compartments (alveolar space, spleen, blood). Incubation of the monocytic cell line THP-1 with LPS or TNF-α resulted in a dose-dependent, significant upregulation of TIE2, suggesting that not recruitment from extra-pulmonary compartments but TIE2 upregulation in resident macrophages accounts for increased lung TEM frequencies.ConclusionsFor the first time, our data provide evidence that the activity of TEMs changes at sites of acute inflammation.

Cerebral Function and Perfusion during Cardiopulmonary Bypass: A Plea for a Multimodal Monitoring Approach

BACKGROUND Postoperative neurological injury still represents a major cause of morbidity after cardiac surgery. Our objective was to compare the limits as well as advantages of routine monitoring tools for the detection of cerebral function and perfusion deficits during cardiopulmonary bypass in a daily clinical setting. METHODS Adult patients undergoing elective cardiac surgery with use of cardiopulmonary bypass were included. Patients received monitoring comprising Bispectral Index (BIS), Near Infrared Spectroscopy (NIRS) and assessment of middle cerebral artery flow velocity (MCAV) using transcranial Doppler (TCD) sonography. Measurements were taken after anesthesia induction (at baseline) and every 10 minutes during aortic cross-clamping. Relative deviation from baseline values was calculated. Values were compared with predefined, generally accepted threshold values identifying patients at risk for cerebral functional and perfusion deficits. RESULTS 30 consecutive patients were included into data analysis. Compared to NIRS as well as BIS monitoring, there was a wide interindividual variability in relative MCAV values for the whole cohort (median 0.9, range 0.39-2.19). Out of 229 measurements in total, 82 BIS but only 30 NIRS and 12 TCD values were lying outside predefined limits. TCD monitoring identified two patients with disturbed cerebral autoregulation, while NIRS remained unremarkable. The latter was significantly associated with systemic hemoglobin levels. Finally, patients with relative MCAV values >1.0 had a higher risk of developing postoperative delirium. CONCLUSION Our findings reveal inherent technical limitations of each individual monitoring component, such as high interindividual variability (TCD), low spatial resolution (NIRS), or interaction with anesthetics (BIS). We therefore argue for a multimodal neuromonitoring that combines several qualities. Such approach would help reducing these limitations while individual components complement each other, thus providing more patient safety during cardiac surgery. Furthermore, such an approach would be easily applicable in a routine clinical setting.