Mechanisms of Pulmonary Endothelial Injury in Sepsis and Critical Illness
CCCF ePoster library. Leligdowicz A. Nov 8, 2018; 233355; 68
Dr. Aleksandra Leligdowicz
Dr. Aleksandra Leligdowicz
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Introduction: The hallmark of septic shock is systemic immune activation, which leads to altered endothelial permeability. The loss of endothelial integrity manifests in vasodilation, microthrombi formation, and vascular leak, which is central to the pathogenesis of the Acute Respiratory Distress Syndrome (ARDS). In vitro methods to study this interplay are limited.


Objectives: Our aim was to optimize an assay to quantify and characterize heterogeneity in inflammation-mediated pulmonary endothelial injury.


Methods: Whole blood was collected from 25 ICU patients with confirmed sepsis from the Early ARDS and Renal Injury (EARLI) cohort, consisting of patients presenting to the emergency department at UCSF-Parnassus with suspected sepsis who were admitted to the ICU, 8 ICU patients from the EARLI cohort without sepsis, and 5 healthy controls. After plasma removal, the cellular layer was diluted with media and stimulated with either LPS or media, followed by centrifugation and supernatant extraction. Human Pulmonary Microvascular Endothelial Cells (HPMECs) were seeded on Electric Cell-substrate Impedance Sensing (ECIS) plates and exposed to culture supernatants. A decrease in resistance signified increased barrier permeability. Flow cytometry was used to assess HPMEC surface markers (ICAM-1, VE-Cadherin, PECAM-1), apoptosis (Annexin V), and viability. Fluorescence microscopy was used to localize surface protein expression and qPCR was used to quantify gene expression (Tie2, Ang1/2, ICAM-1, VE-Cadherin, eNOS).


Results: Heterogeneity was noted in endothelial permeability after exposure to supernatants of LPS-stimulated leukocytes from ICU patients with sepsis. Patients fell into one of 3 groups (Figure 1): no change (10/25, 40%), intermediate change (9/25, 36%), and robust change (6/25, 24%) in permeability. In contrast, ICU patients without sepsis and healthy controls had limited variability in endothelial injury, with patterns similar to the intermediate change group. Change in endothelial permeability was independent of the leukocyte count. When present, an increase in permeability was associated with increased ICAM-1 protein and mRNA expression, and decreased VE-Cadherin, eNOS, and Tie2 mRNA expression (Figure 2).


Conclusion: Supernatants of LPS-stimulated leukocytes derived from septic patients induced pulmonary endothelial permeability with a substantial degree of heterogeneity relative to those derived from critically ill non-septic patients or healthy controls. The heterogeneity in leukocyte response to LPS in these patients may provide insight into why only some patients with septic shock develop acute lung injury. ICAM-1 upregulation on endothelial cells may serve as a positive feedback signal for recruitment of immune cells to the pulmonary vasculature and thereby contribute to inflammation-mediated acute lung injury. Increased permeability was associated with decreased endothelial adhesion and signaling marker expression. This novel assay may be useful for studying mechanisms of pulmonary endothelial injury in critically ill patients.

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