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 “ukhedu”, followed by modifier (“Disgusting”). Sepsis and Pepsis are two forms of biologic breakdown—putrefaction and fermentation, respectively Majno G. JID 1991

Baron RM AJRCMB 2005

Baron RM AJRCMB 2005

Baron RM AJRCMB 2005

Severe Sepsis/ Septic Shock How sepsis kills Severe Sepsis/ Septic Shock Organ Failure Sepsis Hospital mortality Mackenzie I , Lever A BMJ 2007;335:929-932

Acute respiratory distress syndrome (ARDS) Acute onset severe hypoxemia Noncardiogenic, neutrophilic edema High mortality, morbidity Acute respiratory distress syndrome (ARDS) Commonly triggered by sepsis

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 Antithrombin Activated protein C Sepsis remains most common cause of death in ICUs worldwide All failed!

New ideas are needed! New targets New time points

The endothelial glycocalyx Goat coronary capillary van der Berg et al. Circ Res 2003

Glycocalyx and the endothelial surface layer 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

Sepsis and glycocalyx degradation LPS = 20 mg/kg at t = 0 n = 3-6/group

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

Is glycocalyx protection clinically feasible? LPS = 20 mg/kg at t = 0 n = 3-6/group

Importance of glycocalyx recovery? Nothing known!

Sepsis and glycocalyx recovery? LPS = 20 mg/kg at t = 0 n = 3-6/group

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

Our scheme of digital RNA-Seq. (A) General principle of digital RNA-Seq. Assume the original sample contains two cDNA sequences, one with three copies and another with two copies. An overwhelming number of unique barcode sequences are added to the sample in excess, and five are randomly ligated to the cDNA molecules. Ideally, each cDNA molecule in the sample receives a unique barcode sequence. After removing the excess barcodes, the barcoded cDNA molecules are amplified by PCR. Because of intrinsic noise and sequence-dependent bias, the barcoded cDNA molecules are amplified unevenly. Consequently, after the amplicons are sequenced, it appears that there are three copies of cDNA1 for every four copies of cDNA2 based on the relative number of reads for each sequence. However, the ratio in the original sample was 3:2, which is accurately reflected in the relative number of unique barcodes associated with each cDNA sequence. (B) In our implementation of A, we found it advantageous to randomly ligate both ends of each phosphorylated cDNA fragment to a barcoded phosphorylated Illumina Y-shaped adapter. Note that the single T and A overhangs present on the barcodes and cDNA, respectively, are to enhance ligation efficiency. After this step, the sample is amplified by PCR and prepared for sequencing using the standard Illumina library protocol. For each amplicon, both barcode sequences and both strands of the cDNA sequence are read using paired-end deep sequencing. Shiroguchi K et al. PNAS 2012;109:1347-1352 ©2012 by National Academy of Sciences

Our scheme of digital RNA-Seq. (A) General principle of digital RNA-Seq. Assume the original sample contains two cDNA sequences, one with three copies and another with two copies. An overwhelming number of unique barcode sequences are added to the sample in excess, and five are randomly ligated to the cDNA molecules. Ideally, each cDNA molecule in the sample receives a unique barcode sequence. After removing the excess barcodes, the barcoded cDNA molecules are amplified by PCR. Because of intrinsic noise and sequence-dependent bias, the barcoded cDNA molecules are amplified unevenly. Consequently, after the amplicons are sequenced, it appears that there are three copies of cDNA1 for every four copies of cDNA2 based on the relative number of reads for each sequence. However, the ratio in the original sample was 3:2, which is accurately reflected in the relative number of unique barcodes associated with each cDNA sequence. (B) In our implementation of A, we found it advantageous to randomly ligate both ends of each phosphorylated cDNA fragment to a barcoded phosphorylated Illumina Y-shaped adapter. Note that the single T and A overhangs present on the barcodes and cDNA, respectively, are to enhance ligation efficiency. After this step, the sample is amplified by PCR and prepared for sequencing using the standard Illumina library protocol. For each amplicon, both barcode sequences and both strands of the cDNA sequence are read using paired-end deep sequencing. 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 over-represented? Heparan sulfate biosynthesis? Proteoglycans?