Tim Jones

Epigenetic regulatory pathways involving microRNAs may modulate the host immune response following major trauma

BACKGROUND Posttraumatic nosocomial pneumonia is a common complication resulting in significant morbidity. Trauma-induced immunocompromise is associated with an enhanced susceptibility to pneumonia. In this study, we explore the hypothesis that posttranscriptional epigenetic regulation of gene expression may be an important factor in determining this immune phenotype. We describe the pattern of production of microRNAss (miRs) and their association with nosocomial pneumonia following severe trauma. METHODS A convenience sample of 30 ventilated polytrauma patients (UKCRN ID: 5637) and 16 healthy controls were recruited. Messenger RNA and protein levels of key cytokines were quantified within 2 hours of the injury and at 24 hours. Three miRs per cytokine were then selected based on miRBase target prediction scores and quantified using polymerase chain reaction. Nosocomial pneumonia was defined using the Centers for Disease Control and Prevention definitions. RESULTS Median Injury Severity Score (ISS) was 29, and 47% of the patients developed nosocomial pneumonia. miR-125a and miR-202 decreased by 34% and 77%, respectively, immediately following injury, whereas their target, IL-10, increased messenger RNA levels 3-fold and protein levels 180-fold. Tumor necrosis factor &agr; (TNF-&agr;) and IL-12 gene expression decreased by 68% and 43%, respectively, following injury, and this was mirrored by a 10-fold increase in miR-181, an miR predicted to target TNF-&agr; transcripts. Lower levels of miR-125a and miR-374b were associated with the later acquisition of hospital-acquired pneumonia. CONCLUSION Alteration in the expression of miRs with highly predicted complementarity to IL-10 and TNF-&agr; may be an important mechanism regulating the posttraumatic immunosuppressive phenotype in intensive care unit patients. LEVEL OF EVIDENCE Retrospective observational study, level III.

VEGF (Vascular Endothelial Growth Factor) Induces NRP1 (Neuropilin-1) Cleavage via ADAMs (a Disintegrin and Metalloproteinase) 9 and 10 to Generate Novel Carboxy-Terminal NRP1 Fragments That Regulate Angiogenic Signaling

Objective— NRP1(neuropilin-1) acts as a coreceptor for VEGF (vascular endothelial growth factor) with an essential role in angiogenesis. Recent findings suggest that posttranslational proteolytic cleavage of VEGF receptors may be an important mechanism for regulating angiogenesis, but the role of NRP1 proteolysis and the NRP1 species generated by cleavage in endothelial cells is not known. Here, we characterize NRP1 proteolytic cleavage in endothelial cells, determine the mechanism, and investigate the role of NRP1 cleavage in regulation of endothelial cell function. Approach and Results— NRP1 species comprising the carboxy (C)-terminal and transmembrane NRP1 domains but lacking the ligand-binding A and B regions are constitutively expressed in endothelial cells. Generation of these C-terminal domain NRP1 proteins is upregulated by phorbol ester and Ca2+ ionophore, and reduced by pharmacological inhibition of metalloproteinases, by small interfering RNA-mediated knockdown of 2 members of ADAM (a disintegrin and metalloproteinase) family, ADAMs 9 and 10, and by a specific ADAM10 inhibitor. Furthermore, VEGF upregulates expression of these NRP1 species in an ADAM9/10-dependent manner. Transduction of endothelial cells with adenoviral constructs expressing NRP1 C-terminal domain fragments inhibited VEGF-induced phosphorylation of VEGFR2 (VEGF receptor tyrosine kinase)/KDR (kinase domain insert receptor) and decreased VEGF-stimulated endothelial cell motility and angiogenesis in coculture and aortic ring sprouting assays. Conclusions— These findings identify novel NRP1 species in endothelial cells and demonstrate that regulation of NRP1 proteolysis via ADAMs 9 and 10 is a new regulatory pathway able to modulate VEGF angiogenic signaling.

The S100A10 Pathway Mediates an Occult Hyperfibrinolytic Subtype in Trauma Patients

Objective: To determine the characteristics of trauma patients with low levels of fibrinolysis as detected by viscoelastic hemostatic assay (VHA) and explore the underlying mechanisms of this subtype. Background: Hyperfibrinolysis is a central component of acute traumatic coagulopathy but a group of patients present with low levels of VHA-detected fibrinolysis. There is concern that these patients may be at risk of thrombosis if empirically administered an antifibrinolytic agent. Methods: A prospective multicenter observational cohort study was conducted at 5 European major trauma centers. Blood was drawn on arrival, within 2 hours of injury, for VHA (rotation thromboelastometry [ROTEM]) and fibrinolysis plasma protein analysis including the fibrinolytic mediator S100A10. An outcomes-based threshold for ROTEM hypofibrinolysis was determined and patients grouped by this and by D-dimer (DD) levels. Results: Nine hundred fourteen patients were included in the study. The VHA maximum lysis (ML) lower threshold was determined to be <5%. Heterogeneity existed among patients with low ML, with survivors sharing similar clinical and injury characteristics to patients with normal ML values (5–15%). Those who died were critically injured with a preponderance of traumatic brain injury and had a 7-fold higher DD level (died vs. survived: 103,170 vs. 13,672 ng/mL, P < 0.001). Patients with low ML and high DD demonstrated a hyperfibrinolytic biomarker profile, low tissue plasminogen activator levels but high plasma levels of S100A10. S100A10 was negatively correlated with %ML (r = −0.26, P < 0.001) and caused a significant reduction in %ML when added to whole blood ex-vivo. Conclusions: Patients presenting with low ML and low DD levels have low injury severity and normal outcomes. Conversely, patients with low ML but high DD levels are severely injured, functionally coagulopathic and have poor clinical outcomes. These patients have low tissue plasminogen activator levels and are not detectable by ROTEM. S100A10 is a cell surface plasminogen receptor which may drive the hyperfibrinolysis in these patients and which when shed artificially lowers %ML ex-vivo.OBJECTIVE To determine the characteristics of trauma patients with low levels of fibrinolysis as detected by viscoelastic hemostatic assay (VHA) and explore the underlying mechanisms of this subtype. BACKGROUND Hyperfibrinolysis is a central component of acute traumatic coagulopathy but a group of patients present with low levels of VHA-detected fibrinolysis. There is concern that these patients may be at risk of thrombosis if empirically administered an antifibrinolytic agent. METHODS A prospective multicenter observational cohort study was conducted at 5 European major trauma centers. Blood was drawn on arrival, within 2 hours of injury, for VHA (rotation thromboelastometry [ROTEM]) and fibrinolysis plasma protein analysis including the fibrinolytic mediator S100A10. An outcomes-based threshold for ROTEM hypofibrinolysis was determined and patients grouped by this and by D-dimer (DD) levels. RESULTS Nine hundred fourteen patients were included in the study. The VHA maximum lysis (ML) lower threshold was determined to be <5%. Heterogeneity existed among patients with low ML, with survivors sharing similar clinical and injury characteristics to patients with normal ML values (5-15%). Those who died were critically injured with a preponderance of traumatic brain injury and had a 7-fold higher DD level (died vs. survived: 103,170 vs. 13,672 ng/mL, P < 0.001). Patients with low ML and high DD demonstrated a hyperfibrinolytic biomarker profile, low tissue plasminogen activator levels but high plasma levels of S100A10. S100A10 was negatively correlated with %ML (r = -0.26, P < 0.001) and caused a significant reduction in %ML when added to whole blood ex-vivo. CONCLUSIONS Patients presenting with low ML and low DD levels have low injury severity and normal outcomes. Conversely, patients with low ML but high DD levels are severely injured, functionally coagulopathic and have poor clinical outcomes. These patients have low tissue plasminogen activator levels and are not detectable by ROTEM. S100A10 is a cell surface plasminogen receptor which may drive the hyperfibrinolysis in these patients and which when shed artificially lowers %ML ex-vivo.

MicroRNA-mediated regulation of IL-10, IL-12 and TNFα gene expression in severely injured trauma patients

Background Severe trauma induces a blunted immune response associated with an enhanced susceptibility to nosocomial infections [1]. Within 2 hours of injury, expression of the prototypical anti-inflammatory cytokine, IL-10, increases whilst expression of the pro-inflammatory cytokines, TNFa and IL-12, decreases [1]. We hypothesise that microRNAs (miRs) may exacerbate this immunosuppressive gene expression pattern.