1. Using RNA sequencing to investigate 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

2. The ancient riddle of sepsis
Majno G. JID 1991

3. Baron RM AJRCMB 2005

4. Baron RM AJRCMB 2005

5. Baron RM AJRCMB 2005

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

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

8. Have scientific advances translated into clinical benefit?
No therapy targeted at sepsis pathophysiology has ever improved patient survival
Anti-inflammatory therapies
Steroids
Antibodies against inflammatory mediators
Anti-pattern receptor agents
Manipulation of coagulation/inflammation
All failed!

9. New ideas are needed!
New targets
New time points

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

11. Glycocalyx and the endothelial surface layer
ESL: the in vivo glycocalyx
VanTeeffelen et al. Trends Cardiovasc Med 2007

12. 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

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

14. Relevance of ESL to lung inflammation and injury?
VanTeeffelen et al. Trends Cardiovasc Med 2007

15. ESL degradation is necessary for septic ARDS onset in mice
Schmidt et al. Nat Med 2012

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

17. Importance of glycocalyx recovery?
Nothing known!

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

19. Scientific question
What transcriptional events occur within the pulmonary microvasculature during glycocalyx reconstitution?

20. 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

21. 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

22. 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?