Umeå University's logo

Background

Systemic and local tissue hypoxia are contributors to both morbidity and mortality in critical illness and necessitate timely intensive care-based vital organ support. There is a need for better biomarkers to allow early intervention to support recovery for intensive care cases. The aim of this thesis was to first assess factors related to poor outcome in critical care patients with a focus on a regional cohort in the early phase of critical illness, and with a focus on circulatory and respiratory impairment. Then, the aim was to assess early responses to hypoxic and inflammatory stress on a cellular level in lab-based studies, with a focus on extracellular vesicles (EVs) released by cells as potential biomarkers.

Methods

In papers I and II, intensive care cases transported by fixed-wing air ambulance between 2000-2016 were screened for inclusion. A total of 2146 cases were included and all-cause mortality at 90 days post transport was assessed using regression models with relevant co-factors. In Paper III mammalian cells were cultured and EVs were isolated from the conditioned culture media and studied with regard to histone content using an array of molecular biological and biochemical methods including western blotting, immunocytochemistry, transmission electron microscopy and mass spectrometry. In the exploratory study Paper IV, plasma from intensive care cases and healthy volunteers was collected, from which EVs were isolated. Various isolation methods were used and compared, and the EVs were studied with focus on associated histones.

Results

Paper I and II: An increased all-cause mortality risk was found for impaired oxygenation and hemodynamic instability. In addition, the risk of non-daytime transporting of patients was associated with higher mortality risk, not explained by oxygenation or hemodynamic instability. Total transport mission time was assessed for all-cause mortality risk and clinical deterioration during transport. Total mission time was not associated with increased mortality risk. Small negative changes in oxygenation SpO2/FiO2 ratio and mean arterial pressure during transport were observed for the cohort.

Paper III: It was found that histones associated with the outer membrane surface of EVs, and EV secretion increased following cell stress. Histone secretion could be targeted pharmacologically and genetically by targeting intracellular signaling/trafficking pathways and by altering histone posttranslational modifications.

Paper IV: Established methods proved inadequate for reliable EV purification and histone quantification in plasma EV samples. This was likely due to an abundance of plasma proteins co-purifying with EVs, including immunoglobulins. The presence of histones in EVs was confirmed using liquid chromatography mass-spectrometry.

Conclusion

The fixed-wing air ambulance system in Northern Sweden is safe and comparable to other international transport systems, even though the stressors hypoxia and hemodynamic instability were found to contribute to significant all-cause mortality. Histones are associated with EVs and their degree of association can be affected by cell stress, including hypoxia and inflammatory stress. In plasma, improved methods are needed to quantify histone levels in EVs in order to assess their potential use as biomarkers.v

Histones are conserved nuclear proteins that function as part of the nucleosome in the regulation of chromatin structure and gene expression. Interestingly, extracellular histones populate biofluids from healthy individuals, and when elevated, may contribute to various acute and chronic diseases. It is generally assumed that most extracellular histones exist as nucleosomes, as components of extracellular chromatin. We analysed cell culture models under normal and stressed conditions to identify pathways of histone secretion. We report that core and linker histones localize to extracellular vesicles (EVs) and are secreted via the multivesicular body/exosome pathway. Upregulation of EV histone secretion occurs in response to cellular stress, with enhanced vesicle secretion and a shift towards a population of smaller EVs. Most histones were membrane associated with the outer surface of EVs. Degradation of EV-DNA did not impact significantly on EV-histone association. Individual histones  and histone octamers bound strongly to liposomes and EVs, but nucleosomes did not, showing histones do not require DNA for EV binding. Histones colocalized to tetraspanin positive EVs but using genetic or pharmacological intervention, we found that all known pathways of exosome biogenesis acted positively on histone secretion. Inhibition of autophagy and lysosomal degradation had a strong positive effect on EV histone release. Unexpectedly, EV-associated histones lacked the extensive post-translational modification of their nuclear counterparts, suggesting loss of PTMs may be involved in their trafficking or secretion. Our data does not support a significant role for EV-histones existing as nucleosomes. We show for the first time that histones are secreted from cells as membrane proteins via EVs/exosomes. This fundamental discovery provides support for further investigation of the biological activity of exosome associated histones and their role in disease.

Objectives: To test if impaired oxygenation or major haemodynamic instability at the time of emergency intensive care transport, from a smaller admitting hospital to a tertiary care centre, are predictors of long-term mortality.

Design: Retrospective observational study. Impaired oxygenation was defined as oxyhaemoglobin %–inspired oxygen fraction ratio (S/F ratio)<100. Major haemodynamic instability was defined as a need for treatment with norepinephrine infusion to sustain mean arterial pressure (MAP) at or above 60 mm Hg or having a mean MAP <60. Logistic regression was used to assess mortality risk with impaired oxygenation or major haemodynamic instability.

Setting: Sparsely populated Northern Sweden. A fixed-wing interhospital air ambulance system for critical care serving 900 000 inhabitants.

Participants: Intensive care cases transported in fixed-wing air ambulance from outlying hospitals to a regional tertiary care centre during 2000–2016 for adults (16 years old or older). 2142 cases were included.

Primary and secondary outcome measures: All-cause mortality at 3 months after transport was the primary outcome, and secondary outcomes were all-cause mortality at 1 and 7 days, 1, 6 and 12 months.

Results: S/F ratio <100 was associated with increased mortality risk compared with S/F>300 at all time-points, with adjusted OR 6.3 (2.5 to 15.5, p<0.001) at 3 months. Major haemodynamic instability during intensive care unit (ICU) transport was associated with increased adjusted OR of all-cause mortality at 3 months with OR 2.5 (1.8 to 3.5, p<0.001).

Conclusion: Major impairment of oxygenation and/or major haemodynamic instability at the time of ICU transport to get to urgent tertiary intervention is strongly associated with increased mortality risk at 3 months in this cohort. These findings support the conclusion that these conditions are markers for many fold increase in risk for death notable already at 3 months after transport for patients with these conditions.

Objective: We assessed the mortality risk related to the time for intensive care unit transport in a geographically large regional health care system.

Methods: Patient-level data from critical care ambulance missions were analyzed for 2,067 cases, mission time, and relevant patient factors. Mission time was used as a surrogate for the “distance” to tertiary care, and mortality at 7 days and other intervals was assessed.

Results: No increased mortality risk was found at 7 days in an unadjusted regression analysis (odds ratio = 1.00; range, 0.999-1.002; P = .66). In a secondary analysis, an increased mortality risk was observed in longer mission time subgroups and at later mortality assessment intervals (> 375 mission minutes and 90-day mortality; adjusted hazard ratio = 1.56; range, 1.07-2.28; P = .02). Negative changes in oxygenation and hemodynamic status and transport-related adverse events were associated with the longest flight times. Measurable but small changes during flight were noted for mean arterial pressure and oxygenation.

Conclusion: The main finding was that there was no overall difference in mortality risk based on mission time. We conclude that transport distances or accessibility to critical care in the tertiary care center in a geographically large but sparsely populated region is not clearly associated with mortality risk.

The Gram-negative enteropathogen Yersinia pseudotuberculosis possesses a number of regulatory systems that detect cell envelope damage caused by noxious extracytoplasmic stresses. The CpxA sensor kinase and CpxR response regulator two-component regulatory system is one such pathway. Active Cpx signalling upregulates various factors designed to repair and restore cell envelope integrity. Concomitantly, this pathway also down-regulates key determinants of virulence. In Yersinia, cpxA deletion accumulates high levels of phosphorylated CpxR (CpxR~P). Accumulated CpxR~P directly repressed rovA expression and this limited expression of virulence-associated processes. A second transcriptional regulator, RovM, also negatively regulates rovA expression in response to nutrient stress. Hence, this study aimed to determine if CpxR~P can influence rovA expression through control of RovM levels. We determined that the active CpxR~P isoform bound to the promoter of rovM and directly induced its expression, which naturally associated with a concurrent reduction in rovA expression. Site-directed mutagenesis of the CpxR~P binding sequence in the rovM promoter region desensitised rovM expression to CpxR~P. These data suggest that accumulated CpxR~P inversely manipulates the levels of two global transcriptional regulators, RovA and RovM, and this would be expected to have considerable influence on Yersinia pathophysiology and metabolism.

Background Increased endocannabinoid tonus by dual-action fatty acid amide hydrolase (FAAH) and substrate selective cyclooxygenase (COX-2) inhibitors is a promising approach for pain-relief. One such compound with this profile is 2-(2-fluorobiphenyl-4-yl)-N-(3-methylpyridin-2-yl)propanamide (Flu-AM1). These activities are shown by Flu-AM1 racemate, but it is not known whether its two single enantiomers behave differently, as is the case towards COX-2 for the parent flurbiprofen enantiomers. Further, the effects of the compound upon COX-2-derived lipids in intact cells are not known. Methodology/Principal Findings COX inhibition was determined using an oxygraphic method with arachidonic acid and 2-arachidonoylglycerol (2-AG) as substrates. FAAH was assayed in mouse brain homogenates using anandamide (AEA) as substrate. Lipidomic analysis was conducted in unstimulated and lipopolysaccharide + interferon gamma-stimulated RAW 264.7 macrophage cells. Both enantiomers inhibited COX-2 in a substrate-selective and time-dependent manner, with IC50 values in the absence of a preincubation phase of: (R)-Flu-AM1, COX-1 (arachidonic acid) 6 mu M; COX-2 (arachidonic acid) 20 mu M; COX-2 (2-AG) 1 mu M; (S)-Flu-AM1, COX-1 (arachidonic acid) 3 mu M; COX-2 (arachidonic acid) 10 mu M; COX-2 (2-AG) 0.7 mu M. The compounds showed no enantiomeric selectivity in their FAAH inhibitory properties. (R)-Flu-AM1 (10 mu M) greatly inhibited the production of prostaglandin D2 and E2 in both unstimulated and lipopolysaccharide + interferon.-stimulated RAW 264.7 macrophage cells. Levels of 2-AG were not affected either by (R)-Flu-AM1 or by 10 mu M flurbiprofen, either alone or in combination with the FAAH inhibitor URB597 (1 mu M). Conclusions/Significance Both enantiomers of Flu-AM1 are more potent inhibitors of 2-AG compared to arachidonic acid oxygenation by COX-2. Inhibition of COX in lipopolysaccharide + interferon.-stimulated RAW 264.7 cells is insufficient to affect 2-AG levels despite the large induction of COX-2 produced by this treatment.

Histones are conserved nuclear proteins that function as part of the nucleosome in the regulation of chromatin structure and gene expression. Interestingly, extracellular histones populate biofluids from healthy individuals and when elevated may contribute to various acute and chronic diseases. It is generally assumed that most extracellular histones exist as nucleosomes, as components of extracellular chromatin. We analysed cell culture models under normal and stressed conditions to identify pathways of histone secretion. We report that core and linker histones localize to extracellular vesicles (EVs) and are secreted via the multivesicular body/exosome pathway. Upregulation of histone EV secretion occurs in response to cellular stress, with enhanced vesicle secretion and a shift towards a population of smaller EVs. Most histones were membrane associated with the outer surface of EVs. Degradation of EV-DNA did not impact significantly on EV-histone association. Individual histones or histone octamers bound strongly to liposomes and EVs, but nucleosomes did not, showing histones do not require DNA for EV binding. EV histones colocalized most frequently with the tetraspanin CD63 but using genetic or pharmacological intervention, we found that all known pathways of exosome biogenesis acted positively on histone secretion. Inhibition of autophagy and lysosomal degradation had a strong positive effect on EV histone release. Unexpectedly, EV-associated histones lacked the extensive post-translational modification of their nuclear counterparts, suggesting loss of PTMs may be involved in their trafficking or secretion. Our data does not support a significant role for EV-histones existing as nucleosomes. We show for the first time that histones are secreted from cells as membrane proteins via EVs/exosomes. This fundamental discovery provides support for further investigation of the biological activity of exosome associated histones and their role in disease.