1. 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

2. 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

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?
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!

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. RNA quality control
RNA analysis screentape (Agilent): Provides RNA quantification, quality data (“RIN”, 28s/18s)

23. RNA sequencing cDNA library made Coding mRNA isolated RNA fragmented
Reverse transcriptase to cDNA

24. 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:
©2012 by National Academy of Sciences

25. 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:
©2012 by National Academy of Sciences

26. Output?
Changes in entire transcriptome
Splice variants!

27. 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?