Philipp Markart

Extracellular RNA constitutes a natural procoagulant cofactor in blood coagulation

Upon vascular injury, locally controlled haemostasis prevents life-threatening blood loss and ensures wound healing. Intracellular material derived from damaged cells at these sites will become exposed to blood components and could contribute to blood coagulation and pathological thrombus formation. So far, the functional and mechanistic consequences of this concept are not understood. Here, we present in vivo and in vitro evidence that different forms of eukaryotic and prokaryotic RNA serve as promoters of blood coagulation. Extracellular RNA was found to augment (auto-)activation of proteases of the contact phase pathway of blood coagulation such as factors XII and XI, both exhibiting strong RNA binding. Moreover, administration of exogenous RNA provoked a significant procoagulant response in rabbits. In mice that underwent an arterial thrombosis model, extracellular RNA was found associated with fibrin-rich thrombi, and pretreatment with RNase (but not DNase) significantly delayed occlusive thrombus formation. Thus, extracellular RNA derived from damaged or necrotic cells particularly under pathological conditions or severe tissue damage represents the long sought natural “foreign surface” and provides a procoagulant cofactor template for the factors XII/XI-induced contact activation/amplification of blood coagulation. Extracellular RNA thereby reveals a yet unrecognized target for antithrombotic intervention, using RNase or related therapeutic strategies.

Epithelial Endoplasmic Reticulum Stress and Apoptosis in Sporadic Idiopathic Pulmonary Fibrosis

RATIONALE The molecular pathomechanisms underlying idiopathic pulmonary fibrosis (IPF) are elusive, but chronic epithelial injury has recently been suggested as key event. OBJECTIVES We investigated the possible implication of endoplasmic reticulum (ER) stress-mediated apoptosis in sporadic IPF. METHODS We analyzed peripheral explanted lung tissues from patients with sporadic IPF (n = 24), chronic obstructive pulmonary disease (COPD) (n = 9), and organ donors (n = 12) for expression of major ER stress mediators and apoptosis markers by means of immunoblotting, semiquantitative reverse transcription-polymerase chain reaction, immunohistochemistry, and the TUNEL method. MEASUREMENTS AND MAIN RESULTS Compared with COPD and donor lungs, protein levels of ER stress mediators, such as processed p50 activating transcription factor (ATF)-6 and ATF-4 and the apoptosis-inductor CHOP (C/EBP-homologous protein), as well as transcript levels of spliced X-box binding protein (XBP)-1, were significantly elevated in lung homogenates and type II alveolar epithelial cells (AECIIs) of IPF lungs. Proapoptotic, oligomeric forms of Bax, which play a key role in ER stress-mediated apoptosis downstream of CHOP induction, as well as caspase-3 cleavage, could be detected in IPF lungs. By means of immunohistochemistry, exclusive induction of active ATF-6, ATF-4, and CHOP in AECIIs was encountered in IPF but not in COPD or donor lungs. Immunoreactivity was most prominent in the epithelium near dense zones of fibrosis and fibroblast foci, where these ER stress markers colocalized with markers of apoptosis (TUNEL, cleaved caspase-3). CONCLUSIONS Severe ER stress response in the AECIIs of patients with sporadic IPF may underlie the apoptosis of this cell type and development of fibrosis in this disease.

Surfactant alteration and replacement in acute respiratory distress syndrome

The acute respiratory distress syndrome (ARDS) is a frequent, life-threatening disease in which a marked increase in alveolar surface tension has been repeatedly observed. It is caused by factors including a lack of surface-active compounds, changes in the phospholipid, fatty acid, neutral lipid, and surfactant apoprotein composition, imbalance of the extracellular surfactant subtype distribution, inhibition of surfactant function by plasma protein leakage, incorporation of surfactant phospholipids and apoproteins into polymerizing fibrin, and damage/inhibition of surfactant compounds by inflammatory mediators. There is now good evidence that these surfactant abnormalities promote alveolar instability and collapse and, consequently, loss of compliance and the profound gas exchange abnormalities seen in ARDS. An acute improvement of gas exchange properties together with a far-reaching restoration of surfactant properties was encountered in recently performed pilot studies. Here we summarize what is known about the kind and severity of surfactant changes occuring in ARDS, the contribution of these changes to lung failure, and the role of surfactant administration for therapy of ARDS.

Enolase-1 promotes plasminogen-mediated recruitment of monocytes to the acutely inflamed lung.

Cell surface-associated proteolysis plays a crucial role in the migration of mononuclear phagocytes to sites of inflammation. The glycolytic enzyme enolase-1 (ENO-1) binds plasminogen at the cell surface, enhancing local plasmin production. This study addressed the role played by ENO-1 in lipopolysaccharide (LPS)-driven chemokine-directed monocyte migration and matrix invasion in vitro, as well as recruitment of monocytes to the alveolar compartment in vivo. LPS rapidly up-regulated ENO-1 cell-surface expression on human blood monocytes and U937 cells due to protein translocation from cytosolic pools, which increased plasmin generation, enhanced monocyte migration through epithelial monolayers, and promoted matrix degradation. These effects were abrogated by antibodies directed against the plasminogen binding site of ENO-1. Overexpression of ENO-1 in U937 cells increased their migratory and matrix-penetrating capacity, which was suppressed by overexpression of a truncated ENO-1 variant lacking the plasminogen binding site (ENO-1DeltaPLG). In vivo, intratracheal LPS application in mice promoted alveolar recruitment of monocytic cells that overexpressed ENO-1, but not of cells overexpressing ENO-1DeltaPLG. Consistent with these data, pneumonia-patients exhibited increased ENO-1 cell-surface expression on blood monocytes and intense ENO-1 staining of mononuclear cells in the alveolar space. These data suggest an important mechanism of inflammatory cell invasion mediated by increased cell-surface expression of ENO-1.

Disruption of Platelet-derived Chemokine Heteromers Prevents Neutrophil Extravasation in Acute Lung Injury

RATIONALE Acute lung injury (ALI) causes high mortality, but its molecular mechanisms and therapeutic options remain ill-defined. Gram-negative bacterial infections are the main cause of ALI, leading to lung neutrophil infiltration, permeability increases, deterioration of gas exchange, and lung damage. Platelets are activated during ALI, but insights into their mechanistic contribution to neutrophil accumulation in the lung are elusive. OBJECTIVES To determine mechanisms of platelet-mediated neutrophil recruitment in ALI. METHODS Interference with platelet-neutrophil interactions using antagonists to P-selectin and glycoprotein IIb/IIIa or a small peptide antagonist disrupting platelet chemokine heteromer formation in mouse models of ALI. MEASUREMENTS AND MAIN RESULTS In a murine model of LPS-induced ALI, we uncover important roles for neutrophils and platelets in permeability changes and subsequent lung damage. Furthermore, platelet depletion abrogated lung neutrophil infiltration, suggesting a sequential participation of platelets and neutrophils. Whereas antagonists to P-selectin and glycoprotein IIb/IIIa had no effects on LPS-mediated ALI, antibodies to the platelet-derived chemokines CCL5 and CXCL4 strongly diminished neutrophil eflux and permeability changes. The two chemokines were found to form heteromers in human and murine ALI samples, positively correlating with leukocyte influx into the lung. Disruption of CCL5-CXCL4 heteromers in LPS-, acid-, and sepsis-induced ALI abolished lung edema, neutrophil infiltration, and tissue damage, thereby revealing a causal contribution. CONCLUSIONS Taken together, our data identify a novel function of platelet-derived chemokine heteromers during ALI and demonstrate means for therapeutic interference.

Unravelling the progressive pathophysiology of idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a life-threatening condition, with a median survival of <3 yrs. The pathophysiology is not fully understood, but chronic injury of alveolar epithelial type II cells (AECII) is considered key. In IPF, disturbed folding and processing of surfactant proteins and impaired DNA repair may represent underlying reasons for maladaptive endoplasmic reticulum stress responses, increased reactive oxygen species production and/or DNA damage. Excessive AECII apoptosis occurs, leading to permanently perturbed epithelial homeostasis. The role of secondary hits also becomes evident. These may aggravate the disease and result in increased epithelial turnover, exhausting the regenerative capacity of progenitors and disturbing epithelial–mesenchymal interactions. Fibroblast proliferation, transdifferentiation and matrix deposition may be mediated through various mechanisms including epithelial–mesenchymal transition, fibrocyte invasion or expansion of a local fibroblast population. Treatment modalities aiming to attenuate epithelial injury are currently in early pre-clinical development and may reach the clinical arena in only a few years. Meanwhile, novel drugs acting on highly activated fibroblasts such as pirfenidone, an anti-fibrotic drug authorised for IPF in the European Union, or BIBF 1120, a novel triple-kinase inhibitor (blocking vascular endothelial growth factor, platelet-derived growth factor and fibroblast growth factor) currently under clinical investigation, seem to attenuate the progression of IPF.

Safety and tolerability of bosentan in idiopathic pulmonary fibrosis: an open label study

Idiopathic pulmonary fibrosis (IPF) is a fatal disease for which no effective treatment exists. In the present study, 12 IPF patients underwent analysis of gas exchange properties using the multiple inert gas elimination technique on day 1 before and after the administration of 125 mg bosentan, a dual endothelin antagonist. Following this, patients received chronic administration for 12 weeks (62.5 mg b.i.d. in week 1, 125 mg b.i.d. thereafter). The primary objective was to determine the effect of bosentan on gas exchange (day 1) and on oxygen saturation and minute ventilation (week 2). With one exception, where redistribution of total pulmonary blood flow from normal ventilation/perfusion (V′/Q′) areas (93% before, 72% after bosentan) to low V′/Q′ areas (0% before, 22.2% after) was encountered, no patient showed any change in gas exchange (mean±sd shunt flow (% of cardiac output) 8.5±3.4% before, 6.1±2.3% after bosentan; day 1) or oxygen saturation and minute ventilation (week 2). Similarly, none of the secondary parameters was significantly changed either at week 2 or at the end of the study period (week 12). Five patients developed respiratory infections and two died because of pneumonia; this was judged as being unrelated to bosentan intake. In conclusion, bosentan administration does not seem to induce clinically relevant gas exchange abnormalities in idiopathic pulmonary fibrosis patients.

Time-dependent changes in pulmonary surfactant function and composition in acute respiratory distress syndrome due to pneumonia or aspiration

BackgroundAlterations to pulmonary surfactant composition have been encountered in the Acute Respiratory Distress Syndrome (ARDS). However, only few data are available regarding the time-course and duration of surfactant changes in ARDS patients, although this information may largely influence the optimum design of clinical trials addressing surfactant replacement therapy. We therefore examined the time-course of surfactant changes in 15 patients with direct ARDS (pneumonia, aspiration) over the first 8 days after onset of mechanical ventilation.MethodsThree consecutive bronchoalveolar lavages (BAL) were performed shortly after intubation (T0), and four days (T1) and eight days (T2) after intubation. Fifteen healthy volunteers served as controls. Phospholipid-to-protein ratio in BAL fluids, phospholipid class profiles, phosphatidylcholine (PC) molecular species, surfactant proteins (SP)-A, -B, -C, -D, and relative content and surface tension properties of large surfactant aggregates (LA) were assessed.ResultsAt T0, a severe and highly significant reduction in SP-A, SP-B and SP-C, the LA fraction, PC and phosphatidylglycerol (PG) percentages, and dipalmitoylation of PC (DPPC) was encountered. Surface activity of the LA fraction was greatly impaired. Over time, significant improvements were encountered especially in view of LA content, DPPC, PG and SP-A, but minimum surface tension of LA was not fully restored (15 mN/m at T2). A highly significant correlation was observed between PaO2/FiO2 and minimum surface tension (r = -0.83; p < 0.001), SP-C (r = 0.64; p < 0.001), and DPPC (r = 0.59; p = 0.003). Outcome analysis revealed that non-survivors had even more unfavourable surfactant properties as compared to survivors.ConclusionWe concluded that a profound impairment of pulmonary surfactant composition and function occurs in the very early stage of the disease and only gradually resolves over time. These observations may explain why former surfactant replacement studies with a short treatment duration failed to improve outcome and may help to establish optimal composition and duration of surfactant administration in future surfactant replacement studies in acute lung injury.

Mast cells and fibroblasts work in concert to aggravate pulmonary fibrosis: role of transmembrane SCF and the PAR-2/PKC-α/Raf-1/p44/42 signaling pathway.

Mast cell (MC) accumulation has been demonstrated in the lungs of idiopathic pulmonary fibrosis (IPF) patients. Mediators released from MCs may regulate tissue remodeling processes, thereby contributing to IPF pathogenesis. We investigated the role of MC-fibroblast interaction in the progression of lung fibrosis. Increased numbers of activated MCs, in close proximity to fibroblast foci and alveolar type II cells, were observed in IPF lungs. Correspondingly elevated tryptase levels were detected in IPF lung tissue samples. Coculture of human lung MCs with human lung fibroblasts (HLFs) induced MC activation, as evinced by tryptase release, and stimulated HLF proliferation; IPF HLFs exhibited a significantly higher growth rate, compared with control. Tryptase stimulated HLF growth in a PAR-2/PKC-α/Raf-1/p44/42-dependent manner and potentiated extracellular matrix production, but independent of PKC-α, Raf-1, and p44/42 activities. Proproliferative properties of tryptase were attenuated by knockdown or pharmacological inhibition of PAR-2, PKC-α, Raf-1, or p44/42. Expression of transmembrane SCF, but not soluble SCF, was elevated in IPF lung tissue and in fibroblasts isolated from IPF lungs. Coculture of IPF HLFs with MCs enhanced MC survival and proliferation. These effects were cell-contact dependent and could be inhibited by application of anti-SCF antibody or CD117 inhibitor. Thus, fibroblasts and MCs appear to work in concert to perpetuate fibrotic processes and so contribute to lung fibrosis progression.

Current view on alveolar coagulation and fibrinolysis in acute inflammatory and chronic interstitial lung diseases

Acute inflammatory and chronic interstitial lung diseases are characterized by excessive and persistent fibrin deposition in the lung. Intraalveolar fibrin accumulation, observed under these conditions, arises from a leakage of plasma proteins (including fibrinogen) into the alveolar space in combination with a disbalance of alveolar haemostasis. Tissue factor in association with factor VIIa and inhibition of urokinase by plasminogen activator inhibitor-1 are major factors that are responsible for the procoagulant and antifibrinolytic state. In addition, in acute respiratory distress syndrome (ARDS) patients, factor VII-activating protease and extracellular RNA, which may be released into the extracellular milieu from damaged cells during lung injury, may contribute to fibrin formation as well. Fibrin itself can increase vascular permeability, influence the expression of inflammatory mediators and alter the migration and proliferation of various cell types. Additionally, fibrin may inactivate pulmonary surfactant and provide a matrix on which fibroblasts can migrate and produce collagen. Furthermore, cellular activities of haemostatic proteases may also contribute to proinflammatory and fibrotic processes in the lung. The application of coagulation inhibitors, like tissue factor pathway inhibitor, active site-inactivated factor VIIa, activated protein C, antithrombin, heparin or hirudin turned out to be beneficial in experimental models of acute and chronic lung injury. However, the ability of anticoagulant and profibrinolytic agents to improve clinical outcome remains to be elucidated. In the current article, the role of the alveolar coagulation and fibrinolysis systems in acute inflammatory and chronic interstitial lung diseases is discussed with regard to pathomechanisms and modalities of intervention.