Using RNA sequencing to investigate pulmonary endothelial glycocalyx recovery

after sepsis

Eric Schmidt, MDAssistant Professor of Medicine

Pulmonary Sciences and Critical Care MedicineDenver 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

SepsisSevere Sepsis/ Septic Shock

Organ Failure

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

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

Glycocalyx

EC

The endothelial glycocalyx

Goat coronary capillaryvan 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

• Glycocalyx

Measuring the endothelial glycocalyx in mice

Heating plate (titrated to maintain stable rectal temperature)

IV line for continuous anesthetic infusion

IV line for drug, colloid administration

Arterial catheter for MAP, CO monitoring

Ventilator tubing

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SalineLPS

ShamCLP

Sepsis and glycocalyx degradation

LPS = 20 mg/kg at t = 0n = 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

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SalineLPS

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Is glycocalyx protection clinically feasible?

LPS = 20 mg/kg at t = 0n = 3-6/group

Importance of glycocalyx recovery?

Nothing known!

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SalineLPS

ShamCLP

Sepsis and glycocalyx recovery?

LPS = 20 mg/kg at t = 0n = 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

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 over-represented?– Heparan sulfate biosynthesis?– Proteoglycans?