inducing-acute-lung-injury-mice-direct-intratracheal

Inducing Acute Lung Injury in Mice by Direct Intratracheal Lipopolysaccharide Instillation

Airway administration of lipopolysaccharide (LPS) is a common way to study pulmonary inflammation and acute lung injury (ALI) in small animal models. ...

University Hospital Bonn

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. 

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

Cardiac tissue remodeling in the course of chronic left ventricular hypertrophy requires phagocytes which degrade cellular debris, initiate and maintain tissue inflammation and reorganization. The dynamics of phagocytes in left ventricular hypertrophy have not been systematically studied. Here, we characterized the temporal accumulation of leukocytes in the cardiac immune response by flow cytometry and fluorescence microscopy at day 3, 6 and 21 following transverse aortic constriction (TAC). Cardiac hypertrophy due to chronic pressure overload causes cardiac immune response and inflammation represented by an increase of immune cells at all three time points among which neutrophils reached their maximum at day 3 and macrophages at day 6. The cardiac macrophage population consisted of both Ly6C(low) and Ly6C(high) macrophages. Ly6C(low) macrophages were more abundant peaking at day 6 in response to pressure overload. During the development of cardiac hypertrophy the expression pattern of adhesion molecules was investigated by qRT-PCR and flow cytometry. CD11b, CX3CR1 and ICAM-1 determined by qRT-PCR in whole cardiac tissue were up-regulated in response to pressure overload at day 3 and 6. CD11b and CX3CR1 were significantly increased by TAC on the surface of Ly6C(low) but not on Ly6C(high) macrophages. Furthermore, ICAM-1 was up-regulated on cardiac endothelial cells. In fluorescence microscopy Ly6C(low) macrophages could be observed attached to the intra- and extra-vascular vessel-wall. Taken together, TAC induced the expression of adhesion molecules, which may explain the accumulation of Ly6C(low) macrophages in the cardiac tissue, where these cells might contribute to cardiac inflammation and remodeling in response to pressure overload.

Ly6C(low) and not Ly6C(high) macrophages accumulate first in the heart in a model of murine pressure-overload.

The incidence of secondary systemic fungal infections has sharply increased in bacterial septic patients. Antimycotics exhibit immunomodulatory properties, yet these effects are incompletely understood in secondary systemic fungal infections following bacterial sepsis. We investigated a model of systemic inflammation to determine whether antimycotics (liposomal amphotericin B (L-AMB), itraconazol (ITC), and anidulafungin (ANI)) modulate the gene and protein expression as well as the phagocytic activity of lipopolysaccharide (LPS)-stimulated human monocytes.

Antifungal antibiotics modulate the pro-inflammatory cytokine production and phagocytic activity of human monocytes in an in vitro sepsis model.

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.

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

Experimental animal models are indispensable components of preclinical sepsis research. Reproducible results highly rely on defined and invariant baseline conditions. Our hypothesis was that the murine gut microbiota varies among different distributors of laboratory animals and that these variations influence the phenotype of abdominal sepsis derived from a bacterial inoculum model (intraperitoneal stool injection).

Vendor effects on murine gut microbiota influence experimental abdominal sepsis.

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.

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

Breakdown 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.

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

Aneurysmal subarachnoid hemorrhage (aSAH) is a highly complex disease. Majority of aSAH survivors confront post-SAH complications including cerebral vasospasm (CVS) and delayed cerebral ischemia (DCI) that mainly influence the clinical outcome. Tissue damage during early brain injury may lead to release of damage associated molecular pattern molecules (DAMPs) that may initiate and sustain inflammation during the course of aSAH through activation of pattern recognition receptors. Mitochondrial DNA (mtDNA) due to unmethylated CpG motifs acts as a DAMP via binding to toll-like receptor-9. The aim of this study was to investigate the cell free circulating mtDNA in the systemic circulation of aSAH patients and its association with post-SAH complications and clinical outcome. The DNA was extracted from the serum of 80 aSAH patients at days 1, 3, 5, 7, 9, 11, 13 and from 18 healthy controls. Three representative mitochondrial gene fragments including Cytochrome B (CytB), D-Loop and Cytochrome c oxidase subunit-1 (COX-1) were quantified using a Taqman-probes based qPCR. Levels of mtDNA were quantified from standard curves generated using mtDNA extracted from HepG2 cell mitochondria. Clinical outcome of the patients was assessed by Glasgow outcome scale (GOS) and modified Rankin scale (mRS). Clinical data and post-SAH complications were recorded from patient's record file. Serum D-Loop and COX-1 were significantly elevated early after aSAH and remained high over first 2 weeks. CytB levels were however, initially unchanged but elevated later at day 7 as compared to healthy controls. Cumulative levels measured over two weeks showed significant correlations with post-SAH complications including a negative correlation of D-Loop with pneumonia infection, hydrocephalus and occurrence of epilepsy, a positive correlation of Cyt B with occurrence of CVS and a negative correlation of COX-1 with occurrence of systemic infections and seizures. Cumulative D-Loop values negatively correlated with clinical outcome. Our data suggest that mtDNA may directly or indirectly influence post-SAH complications and clinical outcome.

Stilla Frede

Department of Anesthesiology and Intensive Care Medicine

University Hospital Bonn

Inducing Acute Lung Injury in Mice by Direct Intratracheal Lipopolysaccharide Instillation