Hanne Hee Henriksen

A bstract: Pulmonary hypertension (PH) is a pathological condition that affects approximately 1% of the population. The prognosis for many patients is poor, even after treatment. Our knowledge about the pathophysiological mechanisms that cause or are involved in the progression of PH is incomplete. Additionally, the mechanism of action of many drugs used to treat pulmonary hypertension, including sotatercept, requires elucidation. Using our graph-powered knowledge mining software Lifelike in combination with a very small patient metabolite data set, we demonstrate how we derive detailed mechanistic hypotheses on the mechanisms of PH pathophysiology and clinical drugs. In PH patients, the concentration of hypoxanthine, 12(S)-HETE, glutamic acid, and sphingosine 1 phosphate is significantly higher, while the concentration of L-arginine and L-histidine is lower than in healthy controls. Using the graph-based data analysis, gene ontology, and semantic association capabilities of Lifelike , led us to connect the differentially expressed metabolites with G-protein signaling and SRC. Then, we associated SRC with IL6 signaling. Subsequently, we found associations that connect SRC, and IL6 to Activin and BMP signaling. Lastly, we analyzed the mechanisms of action of several existing and novel pharmacological treatments for PH. Lifelike elucidated the interplay between G-protein, interleukin 6, activin, and BMP signaling. Those pathways regulate hallmark pathophysiological processes of PH, including vasoconstriction, endothelial barrier function, cell proliferation, and apoptosis. The results highlight the importance of SRC, ERK1, AKT, and MLC activity in PH. The molecular pathways affected by existing and novel treatments for PH also converge on these molecules. Importantly, sotatercept affects SRC, ERK1, AKT, and MLC simultaneously. The present study shows the power of mining knowledge graphs using Lifelike ’s diverse set of data analytics functionalities for developing knowledge-driven hypotheses on PH pathophysiological and drug mechanisms and their interactions. We believe that Lifelike and our presented approach will be valuable for future mechanistic studies of PH, other diseases, and drugs.

In trauma patients, shock-induced endotheliopathy (SHINE) is associated with a poor prognosis. We have previously identified four metabolic phenotypes in a small cohort of trauma patients (N = 20) and displayed the intracellular metabolic profile of the endothelial cell by integrating quantified plasma metabolomic profiles into a genome-scale metabolic model (iEC-GEM). A retrospective observational study of 99 trauma patients admitted to a Level 1 Trauma Center. Mass spectrometry was conducted on admission samples of plasma metabolites. Quantified metabolites were analyzed by computational network analysis of the iEC-GEM. Four plasma metabolic phenotypes (A–D) were identified, of which phenotype D was associated with an increased injury severity score (p < 0.001); 90% (91.6%) of the patients who died within 72 h possessed this phenotype. The inferred EC metabolic patterns were found to be different between phenotype A and D. Phenotype D was unable to maintain adequate redox homeostasis. We confirm that trauma patients presented four metabolic phenotypes at admission. Phenotype D was associated with increased mortality. Different EC metabolic patterns were identified between phenotypes A and D, and the inability to maintain adequate redox balance may be linked to the high mortality.

Background: Pulmonary hypertension (PH) is classified into five clinical diagnostic groups, including group 1 (idiopathic pulmonary arterial hypertension (IPAH) and connective tissue disease-associated PAH (CTD-aPAH)) and group 4 (chronic thromboembolic pulmonary hypertension (CTEPH)). PH is a progressive, life-threatening, incurable disease. The pathological mechanisms underlying PH remain elusive; recent evidence has revealed that abnormal metabolic activities in the endothelium may play a crucial role. Purpose: Introducing a novel approach for studying PH endothelial function, building on genome-scale metabolic (GEM) reconstruction of the endothelial cell (EC) to investigate intracellular metabolism. Methods: This prospective observational study includes 35 PH patients with either PAH or CTEPH. We have previously developed a platform to study the endothelium cell (EC) by reconstructing the most comprehensive endothelial genome-scale metabolic model (EC-GEM). Since the EC is in direct contact with the blood flow, we can adjust the EC-GEM by measuring extracellular metabolites from the blood and calculating the intracellular metabolic flux in the EC. We conducted individual EC-GEM models, i.e., in total 35 different models. Additionally, each model was widely analyzed; in total, 190 different intracellular activities were investigated. This enables us to develop a heatmap of the intracellular activities at individual patient levels. Patients with similar intracellular endothelial activities were combined. Results: Four distinct intracellular phenotypes were identified; all were independent of the clinical classification of PH. We observed clinically relevant differences in the NT-pro BNP levels and the WHO functional classification between these phenotypes. Also, different important cellular activities were identified separating the four phenotypes. Two phenotypes were characterized by catabolic crisis and extreme citric acid cycle turnover for energy production. However, one of these also suffered from oxidative stress. Conclusion: In PH patients metabolic systems analysis identified four metabolic phenotypes that correlated with NT-pro BNP levels and the WHO functional classification but were independent of the clinical diagnosis. This novel finding may enable us to identify metabolic therapeutic targets specific to the respective phenotypes and thus promote personalized medicine in patients with PH. Funding Acknowledgement: Type of funding sources: Foundation. Main funding source(s): This study was funded by Novo Nordisk Foundation research grant NNF20OC0064556 and the Heart Center Research Council, Copenhagen University Hospital, Rigshospitalet, provided to Jørn Carlsen

Background: The endotheliopathy of trauma (EoT) is associated with increased mortality following injury. Herein, we describe the plasma proteome related to EoT in order to provide insight into the role of the endothelium within the systemic response to trauma. Methods: 99 subjects requiring the highest level of trauma activation were included in the study. Enzyme-linked immunosorbent assays of endothelial and catecholamine biomarkers were performed on admission plasma samples, as well as untargeted proteome quantification utilizing high-performance liquid chromatography and tandem mass spectrometry. Results: Plasma endothelial and catecholamine biomarker abundance was elevated in EoT. Patients with EoT (n = 62) had an increased incidence of death within 24 h at 21% compared to 3% for non-EoT (n = 37). Proteomic analysis revealed that 52 out of 290 proteins were differentially expressed between the EoT and non-EoT groups. These proteins are involved in endothelial activation, coagulation, inflammation, and oxidative stress, and include known damage-associated molecular patterns (DAMPs) and intracellular proteins specific to several organs. Conclusions: We report a proteomic profile of EoT suggestive of a surge of DAMPs and inflammation driving nonspecific activation of the endothelial, coagulation, and complement systems with subsequent end-organ damage and poor clinical outcome. These findings support the utility of EoT as an index of cellular injury and delineate protein candidates for therapeutic intervention.

Disruption to endothelial cell homeostasis results in an extensive variety of human pathologies that are particularly relevant to major trauma. Circulating catecholamines, such as adrenaline and noradrenaline, activate endothelial adrenergic receptors triggering a potent response in endothelial function. The regulation of the endothelial cell metabolism is distinct and profoundly important to endothelium homeostasis. However, a precise catalogue of the metabolic alterations caused by sustained high catecholamine levels that results in endothelial dysfunction is still underexplored. Here, we uncover a set of up to 46 metabolites that exhibit a dose–response relationship to adrenaline-noradrenaline equimolar treatment. The identified metabolites align with the glutathione-ascorbate cycle and the nitric oxide biosynthesis pathway. Certain key metabolites, such as arginine and reduced glutathione, displayed a differential response to treatment in early (4 h) compared to late (24 h) stages of sustained stimulation, indicative of homeostatic metabolic feedback loops. Furthermore, we quantified an increase in the glucose consumption and aerobic respiration in endothelial cells upon catecholamine stimulation. Our results indicate that oxidative stress and nitric oxide metabolic pathways are downstream consequences of endothelial cell stimulation with sustained high levels of catecholamines. A precise understanding of the metabolic response in endothelial cells to pathological levels of catecholamines will facilitate the identification of more efficient clinical interventions in trauma patients.

Introduction: Nearly half of severely injured patients suffer acute kidney injury (AKI), but little is known about its pathogenesis or optimal management. We hypothesized that endothelial dysfunction, evidenced by elevated systemic soluble thrombomodulin (sTM) and syndecan-1, would be associated with higher incidence, worsened severity, and prolonged duration of AKI after severe trauma. Methods: A single-center cohort study of severely injured patients surviving ≥24 h from 2012 to 2016 was performed. Arrival plasma sTM and syndecan-1 were measured by ELISA. Outcomes included 7-day AKI incidence, stage, and prolonged AKI ≥2 days. The Kidney Disease Improving Global Outcomes guidelines were used for AKI diagnosis and staging. Univariate and multivariable analyses were performed. Results: Of 477 patients, 78% were male. Patients had a median age of 38 (interquartile ranges [IQR] 27–54) and injury severity score of 17 (IQR 10–26). AKI developed in 51% of patients. Those with AKI were older and displayed worse arrival physiology. Patients with AKI had higher plasma levels of syndecan-1 (median 34.9 ng/mL vs. 20.1 ng/mL) and sTM (6.5 ng/mL vs. 4.8 ng/mL). After adjustment, sTM and syndecan-1 were both associated with higher AKI incidence, worse AKI severity, and prolonged AKI duration. The strength and precision of the association of sTM and these outcomes were greater than those for syndecan-1. A sensitivity analysis excluding patients with AKI on arrival demonstrated the same relationship. Conclusions: Elevated sTM and syndecan-1, indicating endothelial dysfunction, were associated with higher incidence, worsened severity, and prolonged duration of AKI after severe trauma. Treatments that stabilize the endothelium hold promise for AKI treatment in severely injured patients.

Objective: Investigate the endothelial cell phenotype (s) that causes Shock-Induced Endotheliopathy in trauma. Background: We have studied more than 2750 trauma patients and identified that patients with high circulating syndecan-1 (endothelial glycocalyx damage marker) in plasma have an increased mortality rate compared with patients with lower levels. Notably, we found that patients suffering from the same trauma severity could develop significantly different degrees of endothelial dysfunction as measured by syndecan-1. Methods: Prospective observational study of 20 trauma patients admitted to a Level 1 Trauma Centre and 20 healthy controls. Admission plasma syndecan-1 level and mass spectrometry were measured and analyzed by computational network analysis of our genome-scale metabolic model of the microvascular endothelial cell function. Results: Trauma patients had a significantly different endothelial metabolic profile compared with controls. Among the patients, 4 phenotypes were identified. Three phenotypes were independent of syndecan-1 levels. We developed genome-scale metabolic models representative of the observed phenotypes. Within these phenotypes, we observed differences in the cell fluxes from glucose and palmitate to produce Acetyl-CoA, and secretion of heparan sulfate proteoglycan (component of syndecan-1). Conclusions: We confirm that trauma patients have a significantly different metabolic profile compared with controls. A minimum of 4 shock-induced endotheliopathy phenotypes were identified, which were independent of syndecan-1level (except 1 phenotype) verifying that the endothelial response to trauma is heterogeneous and most likely driven by a genetic component. Moreover, we introduced a new research tool in trauma by using metabolic systems biology, laying the foundation for personalized medicine.

Severe trauma is accompanied by endothelial glycocalyx disruption, which drives coagulopathy, increasing transfusion requirements and death. This syndrome has been termed endotheliopathy of trauma (EOT). Some have suggested EOT results from endothelial cellular damage and apoptosis. Endothelial microvesicles (EMVs) represent cellular damage. We hypothesized that EOT is associated with endothelial damage and apoptosis resulting in an increase in circulating EMVs. Prospective, observational study enrolling severely injured patients. Twelve patients with EOT, based on elevated Syndecan-1 levels, were matched with 12 patients with lower levels, based on Injury Severity Score (ISS), abbreviated injury scale profile, and age. Thrombelastography and plasma levels of biomarkers indicative of cellular damage were measured from blood samples collected on admission. EMVs were determined by flow cytometry using varied monoclonal antibodies associated with endothelial cells. Significance was set at P < 0.05. Admission physiology and ISS (29 vs. 28) were similar between groups. Patients with EOT had higher Syndecan-1, 230 (158, 293) vs. 19 (14, 25) ng/mL, epinephrine, and soluble thrombomodulin levels. Based on thrombelastography, EOT had reductions in clot initiation, amplification, propagation and strength, and a greater frequency of transfusion, 92% vs. 33%. There were no differences in EMVs irrespective of the antibody used. Plasma norepinephrine, sE-selectin, sVE-cadherin, and histone-complexed DNA fragments levels were similar. In trauma patients presenting with EOT, endothelial cellular damage or apoptosis does not seem to occur based on the absence of an increase in EMVs and other biomarkers. Thus, this suggests endothelial glycocalyx disruption is the underlying primary cause of EOT.