using rna sequencing to investigate glycocalyx recovery after sepsis eric schmidt, md assistant...
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Using RNA sequencing to investigate 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?
• 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!
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
0
0.5
1
1.5
2
2.5
3
0 0.5 1 1.5 24 72
ES
L th
ickn
ess
(µm
)
Time (hours)
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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0.5
1
1.5
2
2.5
3
0 0.5 1 1.5 24 72
ES
L th
ickn
ess
(µm
)
Time (hours)
SalineLPS
ShamCLP
Is glycocalyx protection clinically feasible?
LPS = 20 mg/kg at t = 0n = 3-6/group
Importance of glycocalyx recovery?
Nothing known!
0
0.5
1
1.5
2
2.5
3
0 0.5 1 1.5 24 72
ES
L th
ickn
ess
(µm
)
Time (hours)
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
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?