Download MicroRNAs in Ex Vivo Stored Blood Cells

MicroRNAs in Ex Vivo Stored Blood Cells:
What Future Do They Hold in Transfusion
Medicine?
C.D. Atreya, Ph.D.
Associate Director for Research
Office of Blood Research and Review
Center for Biologics Evaluation and Research
US Food and Drug Administration
US Department of Health and Human Services
5th World Hematologists Congress, Aug 18-19, 2016
***This presentation reflects the views of Dr. C.D. Atreya and should
not be construed to represent FDA’s views or policies***
 MicroRNAs
Small Single-stranded noncoding regulatory RNAs
which represent ~4% of the genes in the human
genome
 Ex Vivo Stored Blood Cells
Platelets and RBCs collected, processed, and stored in
bags as PC and pRBC units available for transfusion
 Transfusion Medicine
A branch of medicine that is concerned with safe and
effective transfusion of blood and blood components
(e.g. Platelets and RBC)
Topics covered….
 miRNAs –Biogenesis and mechanism of action
 Changes associated with ex vivo Storage of
blood Cells (PCs and pRBC)
 Roll of miRNAs in stored blood cells
 Extracellular release of miRNAs in the context
of ex vivo stored blood cells
 Conclusions
 miRNAs –Biogenesis and mechanism of action
 Changes associated with ex vivo Storage of
blood Cells (PCs and pRBC)
 Roll of miRNAs in stored blood cells
 Extracellular release of miRNAs in the context
of ex vivo stored blood cells
 Conclusions
http://www.sigmaaldrich.com/
MicroRNA (miRNA)
Biogenesis and mechanism of action
 Single-stranded small noncoding regulatory RNAs
 Represent ~4% of the genes in the human genome
Drsha
Nuclear RNAse III
Dicer
Cytoplasmic RNAse III
DGCR8 protein
Digeorge Syndrome Critical Region 8
RISC
RNA induced silence complex
seed
Single miRNA can control multiple mRNAs
Multiple miRNAs can effectively control a
single mRNA by targeting at different sites
 miRNAs –Biogenesis and mechanism of action
 Changes associated with ex vivo Storage of
blood Cells (PCs and pRBC)
 Roll of miRNAs in stored blood cells
 Extracellular release of miRNAs in the context
of ex vivo stored blood cells
 Conclusions
Blood Cell Lineage
= Platelets
No nucleus!
RBC
No nucleus!
Model of miRNA pathway
in megakaryocyteerythrocyte progenitor
cells (MEPs) and
anucleate blood cells
Ryan and Atreya 2011
Transfusion Med Reviews , 25:247-251
Stored blood cells
1. Packed RBC
Ex vivo storage @ 1-6 0C with
certain anti-coagulants for 42 days;
Can be stored @-65 0C in glycerol
by specified methods
2. Platelet Concentrates
Ex vivo storage @ Controlled
Room temp (20-24 0C)
RBC ex vivo storage
 Currently licensed RBC additive solutions (AS) have
acidic pH (~5.5-5.8)
 The acidic intracellular environment rapidly decreases
ATP (necessary for RBC survival) and 2,3-DPG
(necessary for oxygen delivery) and triggers apoptosislike processes (eryptosis)
 Research on RBC storage has repeatedly demonstrated
that fundamental biology of RBC is still not well
understood
Sparrow, Blood Transfusion 2012
RBC Storage Lesion (SL)
“Biochemical, morphological and immunologic changes
which occur within RBC and in the associated storage media
during ex vivo preservation of RBC are collectively known
as RBC storage lesion”
Biochemical: rapid depletion of 2, 3-diphosphoglycerate (DPG) and
Adenosine triphosphate (ATP) levels in RBC
Morphological: Change in corpuscle shape affecting microcirculation in
the recipients
Immunological: Immuno-modulatory effects in the recipients
Why Study RBC-SL?
Ex Vivo Stored RBC with SL
Clinical consequences
Biochemical
morphological
immunologic
changes
Acute lung injury
Multi-organ failure
Mortality
Modified Fig.1 from Kor et al, BJBMC 2009
Human RBC
What we know
1. Mature RBC is enucleated
2. During ex vivo storage, RBC undergo changes (SL)
3. RBC-SL has serious clinical consequences
What we didn’t know
1. How are the cellular processes regulated in enucleated RBC,
especially in RBC-SL?
2. Are there other ‘command & controls’ to compensate for the
loss of nucleus in mature RBC?
2. Can we manipulate these controls to improve the quality and
shelf-life of RBC in storage?
Topics covered….
 miRNAs –Biogenesis and mechanism of action
 Changes associated with ex vivo Storage of
blood Cells (PCs and pRBC)
 Roll of miRNAs in stored blood cells
 Extracellular release of miRNAs in the context
of ex vivo stored blood cells
 Conclusions
Recent research advances in RBC and platelets
 Enucleated mature RBC and platelets have mRNAs, splicing factors
etc. in addition to proteins (Denis et al, 2005)
 Seminal observations are, RBC and platelets have abundant and
diverse array of microRNAs (Landry et al, 2009; Chen et al, 2009;
Kannan et al 2009; Kannan and Atreya, 2010; Sarachana et al 2015;
Dahiya et al 2016)
 MicroRNAs (miRNAs) are established as the key cellular negative
regulators of genes (mRNAs) in eukaryotic cells
 In enucleated blood cells by far the miRNA is the only major
regulatory nucleic acid identified that can regulate cellular processes!
 Therefore, understanding the blood cell SL processes through miRNA
regulation in ex vivo storage will provide clues towards enhancing the
quality and perhaps shelf-life of these cells during storage
Understanding the regulatory mechanisms of RBC
storage lesion
Some of the measurable parameters during storage
RBC shape change (affects microcirculation) as
measured by Mean Corpuscular Volume (MCV)
Cellular ATP loss
Apoptosis-like (eryptosis) symptoms
miRNA analysis in stored red blood cells
Study design and methods
• Leukocyte-reduced pRBC units collected from 11 donors were stored
under conventional blood bank conditions
• Samples were collected from each bag at day 0, 7, 14, 28, 42, and
56 and mature RBCs were enriched and isolated
• Three known RBC-SL parameters were chosen for the study [Mean
Corpuscular Volume (MCV), eryptosis and ATP loss]
• Purified RNA from each mature RBC sample was subjected to highthroughput miRNA microarray* differential expression profiling and
the data was subjected to various bioinformatics programs
*Affymetrix GeneChip miRNA 3.0 microarrays representing 19,724
probes covering over 5,600 miRNAs, pre-miRNAs, snoRNAs etc.
Changes in RBC-SL parameters during storage
Increased MCV
ATP loss
Sarachana et al, Transfusion 2015
Eryptosis
Normalized microarray data set was uploaded into the TMeV program* and
Pavlidis Template Matching (PTM) analysis was performed using the level of
eryptosis, ATP or Mean Corpuscular Volume (MCV) as the template for correlation
analysis.
92 common miRNAs
Correlates to all
3 RBC-SL parameters
MCV
ATP
Eryptosis
* TIGR Multiple Array Viewer software package (TMeV version 3.0)
miR-196a and miR-1269
were differentially
expressed over time
Sarachana et al, Transfusion 2015
Top two cellular functions associated with potential
targets of miR-196a and miR-1269 were identified
Identified Function
P-value
No. of genes (mRNAs)
Cell morphology
Fisher’s exact test,
<0.05
74
Cell death and survival
Fisher’s exact test,
<0.05
97
These data were obtained using Ingenuity Pathway Analysis (IPA) software.
P-values were calculated using Fisher’s exact test, which was performed
using the entire set of genes within the Ingenuity Knowledge Base as the
reference set.
We selected miR-196a and evaluated its effects on cell death
and/or survival
Sarachana et al, Transfusion 2015
Overexpression of hsa-miR-196a reduces early programmed cell death and
enhances ATP concentration in a human erythroblast cell line (HEL92.1.7)
This suggests that miR-196a has the
potential to slow eryptosis in stored RBC
Sarachana et al, Transfusion 2015
miRNA analysis in stored red blood cells
Summary
 Our analysis identified two RNAs (miR-196a and miR-1269) whose
changes in the expression levels were correlated with the
selected three SL parameters that we evaluated.
 Overexpression of one such miRNA, the miR-196a, in a human
erythroblast cell line (HEL 97.1.2) confirmed its protective
effects against cell death and ATP loss.
In support of our concept that miRNAs can protect RBC from eryptosis, Yu et al,
2010 have demonstrated that miR-451 protects erythrocytes against oxidant
stress (Yu et al, Genes and Development, 2010, 24:1620-33)
Platelet Storage Lesion (PSL)
Shrivastava, 2009. Transfusion and Apheresis Science 41:105-113.
Significant mRNAs P<0.01,
fdr<0.05
Significant miRNAs P<0.05
Downregulated mRNA
targets
Upregulated mRNA
targets
Upregulated miRNAs
miRNA target filter
analysis using IPA
Downregulated miRNAs
Platelet miRNA and mRNA differential analysis
(Day5 vs. Day9 relative to Day0 of storage)
IPA functions, pathways,
network
mRNA target enrichment
analysis for selected mRNAs
(704 mRNA targets selected
based on opposite correlation
with miRNAs)
mRNA:miRNA
correlation based on
opposite expression
using IPA using
experimental target set
144 differentially expressed
miRNAs and 704 differentially
expressed mRNA targets forming
5261 miRNA:mRNA pairs
Dahiya et al, Platelets 2016 (in press)
miR-570 expression
in platelets (Plts)
ATP5L mRNA levels in
miR-570 expressed Plts
Two miR-570 target sites
in ATP5L mRNA 3’UTR
ATP levels in miR-570
expressed Plts
Low levels of miR-570 can be a
predictive quality biomarker
for stored platelets
Dahiya et al, Platelets 2016 (in press)
Another example of miRNAs in stored
platelets
Our miRNA analysis (Day 0-Day 9 of storage) identified
Ras-related protein1 (Rap1) signaling pathway as one of
the potential miRNA targets in stored platelets
It is known that during platelet storage Rap1 increases and
triggers platelet activation as monitored by Flow Cytometry
using the activation marker CD62P (Shubert et al, Transfusion
2009)
Testable Hypothesis: RAP1 (mRNA) down-regulation by miRNAs
should suppress platelet activation during storage and provide
opportunities to improve platelet quality in storage
Dahiya et al, Unpublished data
Ground work
By using Yoon et al, 2012
method, we have enriched
20 miRNAs that are tightly
associated with RAP1
mRNA 3’UTR
RAP 1 expression in megakaryocyte cells
is suppressed by miR-181, miR-202 and
miR-320
From this list, 5 randomly
selected miRNAs were
introduced into a human
megakaryocyte cell line and
after 48 h, evaluated their
effect on RAP1 expression
Dahiya et al, Unpublished data
Topics covered….
 miRNAs –Biogenesis and mechanism of action
 Changes associated with ex vivo Storage of
blood Cells (PCs and pRBC)
 Roll of miRNAs in stored blood cells
 Extracellular release of miRNAs in the context
of ex vivo stored blood cells
 Conclusions
Extracellular release of miRNAs
Taken up by other cells/
degraded/excreted
Packaged in
exosomes
RNA pol II/III
Export thru’ RNA binding
proteins
Export thru’
microvesicles
During
membrane
blebbing
Etheridge et al, Mutat Res 2011, 717:85-90
Mechanism of miRNA release from cells
in the peripheral blood
(Ago2=Argonaute2; NPM1=nucleophosmin1; HDL=high density lipoproteins)
Stefano et al, Vascul Pharmacol 2011 Oct;55(4):111-8. Epub 2011 Aug 6.
 Platelet MP-derived miR-223 can regulate human
umbilical vein endothelial cells (HUVEC) gene
expression at the protein level
 Regulates FBXW7 (a F-Box protein, subunit of ubiquitin
protein)
 Regulates EFNA1 protein, a member of ephrin family of
proteins
 Platelet MPs (containing miRs, e.g. miR-126-3p)
reprogram macrophage gene expression and function
 Platelet MPs induced up-regulation of 34 miRs and
downregulation of 367 mRNAs
Laffont et al, Blood 2013, 122:253-261; Thrombosis and Haemostasis 2016, 115:311-323.
Topics covered….
 miRNAs –Biogenesis and mechanism of action
 Changes associated with ex vivo Storage of
blood Cells (PCs and pRBC)
 miRNAs’ Roll in stored blood cells
 Extracellular release of miRNAs in the context
of ex vivo stored blood cells
 Conclusions
MicroRNAs in Stored Blood Cells: What Future Do
They Hold in Transfusion Medicine?
 miRNAs regulate physiological events of mature (enucleated) blood
cells
 reversing this regulation may help enhance their quality and
shelf-life during storage
 some of the miRNAs could serve as quality biomarkers of storage
 Enhancing the quality and shelf-life of stored blood cells will
increase inter-donation intervals for repeat donors, especially for
women whose iron deficiencies can be mitigated by long interdonation intervals!
 Blood cell-derived MP miRNAs can regulate gene expression of other
cells
 Can transfusion of ex vivo stored blood cells with miRNA
containing MPs develop measurable transient alteration of gene
expression in other cells of recipients?
Enhancing the quality and shelf-life of stored blood cells will increase
inter-donation intervals for repeat donors, especially for women whose
iron deficiencies can be mitigated by long inter-donation intervals!
Advancing innovation is fundamental to US FDA’s
core mission of protecting and promoting the public
health
-FDA’s Regulatory Science Priorities (2014-2018)
Thank you!