Using RNA sequencing to investigate pulmonary endothelial glycocalyx recovery after sepsis Eric Schmidt, MD Assistant Professor of Medicine Pulmonary Sciences and Critical Care Medicine Denver Health Medical Center University of Colorado School of Medicine The ancient riddle of sepsis Majno G. JID 1991 Baron RM AJRCMB 2005 Baron RM AJRCMB 2005 Baron RM AJRCMB 2005 How sepsis kills Sepsis Organ Failure Mackenzie I , Lever A BMJ 2007;335:929-932 Severe Sepsis/ Septic Shock Hospital mortality Acute onset severe hypoxemia Noncardiogenic, neutrophilic edema High mortality, morbidity Commonly triggered by sepsis Acute respiratory distress syndrome (ARDS) Have scientific advances translated into clinical benefit? • Many therapies have been developed to target sepsis pathophysiology – Anti-inflammatory therapies • Corticosteroids • Antibodies against inflammatory mediators • Anti-pattern receptor agents – Manipulation of coagulation/inflammation All failed! • Antithrombin • Activated protein C • Sepsis remains most common cause of death in ICUs worldwide New ideas are needed! • New targets • New time points The endothelial glycocalyx Glycocalyx EC Goat coronary capillary van der Berg et al. Circ Res 2003 Glycocalyx and the endothelial surface layer • Glycocalyx • ESL: the in vivo glycocalyx VanTeeffelen et al. Trends Cardiovasc Med 2007 Measuring the endothelial glycocalyx in mice Ventilator tubing Heating plate (titrated to maintain stable rectal temperature) Arterial catheter for MAP, CO monitoring IV line for drug, colloid administration IV line for continuous anesthetic infusion ESL thickness (µm) Sepsis and glycocalyx degradation Sham CLP Saline LPS 3 2.5 2 1.5 1 0.5 0 0 LPS = 20 mg/kg at t = 0 n = 3-6/group 0.5 1 1.5 24 Time (hours) 72 Relevance of ESL to lung inflammation and injury? VanTeeffelen et al. Trends Cardiovasc Med 2007 ESL degradation is necessary for septic ARDS onset in mice Schmidt et al. Nat Med 2012 ESL thickness (µm) Is glycocalyx protection clinically feasible? Sham CLP Saline LPS 3 2.5 2 1.5 1 0.5 0 0 LPS = 20 mg/kg at t = 0 n = 3-6/group 0.5 1 1.5 24 Time (hours) 72 Importance of glycocalyx recovery? Nothing known! ESL thickness (µm) Sepsis and glycocalyx recovery? Sham CLP Saline LPS 3 2.5 2 1.5 1 0.5 0 0 LPS = 20 mg/kg at t = 0 n = 3-6/group 0.5 1 1.5 24 Time (hours) 72 Scientific question • What transcriptional events occur within the pulmonary microvasculature during glycocalyx reconstitution? Modeling septic lung injury in mice • Cecal ligation and puncture – “Gold standard” for polymicrobial sepsis (Rittirsch et al. Nat Protocols 2009) – Lung injury may be augmented by moderate (60%) hyperoxia (Aggarwal et al. AJP-Lung 2010) • “Double-hit” model with clinical relevance Lung harvest and RNA extraction • 48 hours after CLP/hyperoxia: – Mice euthanized – Pulmonary artery flushed with RNAlater • Periphery of lung harvested (endothelium-rich) • Kept in RNAlater overnight at 4 degrees C • Controls: sham/hyperoxia x 48 hours RNA quality control RNA analysis screentape (Agilent): Provides RNA quantification, quality data (“RIN”, 28s/18s) RNA sequencing • cDNA library made – Coding mRNA isolated – RNA fragmented – Reverse transcriptase to cDNA Shiroguchi K et al. PNAS 2012;109:1347-1352 ©2012 by National Academy of Sciences Shiroguchi K et al. PNAS 2012;109:1347-1352 ©2012 by National Academy of Sciences Output? • Changes in entire transcriptome • Splice variants! Analysis expectations • Unbiased list of transcripts that are differentially expressed in CLP and sham – Hypothesis-generating • Are certain biological pathways overrepresented? – Heparan sulfate biosynthesis? – Proteoglycans?