using rna sequencing to investigate pulmonary endothelial glycocalyx recovery after sepsis eric...
TRANSCRIPT
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
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!
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
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2.5
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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
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
Is glycocalyx protection clinically feasible?
LPS = 20 mg/kg at t = 0n = 3-6/group
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
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