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Peder S Olofsson1,2, Benjamin E Steinberg2,3, Roozbeh Sobbi4, Maureen A Cox3, Mohamed N Ahmed5,
Michaela Oswald6, Ferenc Szekeres7,8, William M Hanes2, Andrea Introini9, Shu Fang Liu5,
Nichol E Holodick10, Thomas L Rothstein10, Cecilia Lvdahl7, Sangeeta S Chavan2, Huan Yang2,
Valentin A Pavlov2, Kristina Broliden9, Ulf Andersson11, Betty Diamond12, Edmund J Miller5,
Anders Arner7, Peter K Gregersen6, Peter H Backx4,13, Tak W Mak3 & Kevin J Tracey2,11
Blood pressure regulation is known to be maintained by a
neuro-endocrine circuit, but whether immune cells contribute
to blood pressure homeostasis has not been determined.
We previously showed that CD4+ T lymphocytes that express
choline acetyltransferase (ChAT), which catalyzes the synthesis
of the vasorelaxant acetylcholine, relay neural signals1.
Here we show that these CD4+CD44hiCD62Llo T helper cells
by gene expression are a distinct T-cell population defined by
ChAT (CD4 TChAT). Mice lacking ChAT expression in CD4+ cells
have elevated arterial blood pressure, compared to littermate
controls. Jurkat T cells overexpressing ChAT (JTChAT) decreased
blood pressure when infused into mice. Co-incubation of
JTChAT and endothelial cells increased endothelial cell levels
of phosphorylated endothelial nitric oxide synthase, and of
nitrates and nitrites in conditioned media, indicating increased
release of the potent vasorelaxant nitric oxide. The isolation
and characterization of CD4 TChAT cells will enable analysis of
the role of these cells in hypotension and hypertension, and
may suggest novel therapeutic strategies by targeting cellmediated vasorelaxation.
A major physiological mechanism of blood pressure regulation is
through modulation of blood flow by alterations in vessel diameter.
Acetylcholine, a signaling molecule produced by neurons and lymphocytes, is a vasorelaxant that decreases blood vessel resistance and
reduces blood pressure2. Binding to cognate cholinergic receptors on
endothelial cells, acetylcholine stimulates phosphorylation of endothelial nitric oxide synthase (eNOS), the rate-limiting enzyme in the
biosynthesis of nitric oxide (NO). Endothelium-derived NO diffuses
into smooth muscle cells in the vascular wall, where it interacts with
1Center
for Bioelectronic Medicine, Department of Medicine, Center for Molecular Medicine, Solna, Karolinska Institutet, Karolinska University Hospital, Stockholm,
Sweden. 2Laboratory of Biomedical Science, The Feinstein Institute for Medical Research, Manhasset, New York, USA. 3The Campbell Family Institute for Breast
Cancer Research, University Health Network, Toronto, Ontario, Canada. 4Division of Cardiology, Peter Munk Cardiac Centre, University Health Network, Toronto,
Ontario, Canada. 5Center for Heart and Lung Research, The Feinstein Institute for Medical Research, Manhasset, New York, USA. 6Robert S. Boas Center for
Genomics and Human Genetics, Feinstein Institute for Medical Research, Manhasset, New York, USA. 7Department of Physiology and Pharmacology, Karolinska
Institutet, Stockholm, Sweden. 8School of Health and Education, University of Skvde, Skvde, Sweden. 9Department of Medicine, Solna, Unit of Infectious Diseases,
Center for Molecular Medicine, Karolinska Institutet, Karolinska University Hospital, Stockholm, Sweden. 10Center for Oncology and Cell Biology, The Feinstein
Institute for Medical Research, Manhasset, New York, USA. 11Department of Womens and Childrens Health, Karolinska Institutet, Stockholm, Sweden. 12The Center
for Autoimmune and Musculoskeletal Diseases, The Feinstein Institute for Medical Research, Manhasset, New York, USA. 13Department of Biology, York University,
Toronto, Ontario, Canada. Correspondence should be sent to P.S.O. (peder.olofsson@ki.se).
Received 6 June; accepted 4 August; published online 12 September 2016; doi:10.1038/nbt.3663
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Figure 1 Gene expression analysis reveals differences between CD4 +CD44hiCD62Llo ChAT-eGFP+ and ChAT-eGFP T cells. (a) RNA from the ChATeGFP+ and ChAT-eGFP subsets of CD4+CD44hiCD62Llo T cells was analyzed by Affymetrix Gene ST 1.0 arrays. Unsupervised hierarchical clustering of
the whole transcriptome of six samples, each from 3 pooled murine spleens, is shown. (b) The 1,288 transcripts with significantly different expression
between the subsets (P < 0.05 with BenjaminiHochberg correction) were studied using unsupervised hierarchical clustering and plotted in a heat map.
1,281 out of 1,288 transcripts had a fold change of >1.5 (n = 3). (c) Bottom: unsupervised global hierarchical clustering of gene expression in
ChAT-eGFP+ T lymphocytes and 198 other immune cell subsets from the ImmGen data set. Top: expression level of ChAT across the 199 different
immune cell subsets. Bar color indicates cell type as shown in the key. Gene expression in CD4 +CD44hiCD62Llo ChAT-eGFP+ T cells was analyzed in
the context of 13 other subsets isolated from spleen in the ImmGen data set. (d) Unsupervised hierarchical clustering of gene expression in the T-cell
subsets derived from spleen. (e,f) Heat map of pair-wise Euclidian distances (green color indicates short distance and white color long distance) (e) and
principal component analysis of gene expression in the splenic T-cell subsets (f).
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tool (GOrilla)9 revealed that genes modulating immune process regulation and negative regulation of leukocyte activation were highly
overrepresented (Supplementary Fig. 1); and genes modulating
G-protein-coupled signaling were downregulated (Supplementary
Fig. 2) in the ChAT-eGFP+ population. Analysis using the Kyoto
Encyclopedia of Genes and Genomes (KEGG) database by the DAVID
Bioinformatics tools1012 showed over-representation of genes implicated in cytokine-cytokine receptor interaction (Supplementary
Table 1) in the ChAT-eGFP+ population. ChAT expression in ChATeGFP+ T lymphocytes was compared against that in 198 different
immune cell subsets in the ImmGen data set13 (cell types listed in
Supplementary Table 2). The ChAT-eGFP+ T-cell subset expressed
ChAT at considerably higher levels as compared to the other subsets (Fig. 1c and Supplementary Table 3). Microarray analysis using
unsupervised hierarchical clustering demonstrated that ChAT-eGFP+
T lymphocytes clustered with CD4+ CD44hi and Foxp3-expressing
T lymphocytes (Fig. 1c).
ChAT-eGFP+ T cells within the CD4+CD44hiCD62Llo T helper cell
population defined a unique branch, distinct from other splenic T lymphocyte subsets in hierarchical clustering (Fig. 1d). Pairwise Euclidean
distance plots of complete gene expression revealed that ChAT-eGFP+
T lymphocytes were highly segregated (Fig. 1e). Principal component analysis showed that ChAT-eGFP+ T lymphocytes harbored a
T-cell gene signature (Supplementary Fig. 3 and Supplementary
Table 4), but differed as compared to other T lymphocyte subsets
by the first principal component (Fig. 1f). Gene ontology classification demonstrated that genes implicated in regulation of leukocyte
activation, cytokine receptor activity, and G-protein-coupled receptor
activity were highly over-represented (Supplementary Figs. 4 and 5).
P = 0.01
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ChATexpressing
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Figure 2 Increased blood pressure in mice with genetic ablation of choline acetyltransferase + CD4+ cells. (a) Histogram of ChAT-eGFP+ cells in the CD3+
cell population in blood obtained by tail bleed from male and female ChAT-eGFP reporter mice at 612 weeks of age analyzed by flow cytometry.
(b) Graphs of blood pressure measured by telemetry over 48 h in awake 12-week-old ChAT-deficient mice (n = 6) and their ChAT-expressing (n = 4)
littermate controls. Data were analyzed using repeated measures ANOVA. Open circles, littermate controls, filled circles, ChAT deficient. Error bars, s.e.m.
(c) Blood pressure (BP) as measured by an indwelling catheter in the left carotid artery in anesthetized ChAT-expressing (+; n = 3) and ChAT-deficient (;
n = 4) mice. D, diastolic; M, mean arterial; S, systolic. *P < 0.05 (two-tailed Students t test). Open symbols, littermate controls, filled symbols, ChAT
deficient. (d) Echocardiographic measurements in male 10- to 11-week- old ChAT-expressing (n = 6) and ChAT-deficient (n = 5) mice. CO, cardiac output;
SV, stroke volume; FS, fractional shortening; EF, ejection fraction. *P < 0.05, ***P < 0.001; n.s., not significant (two-tailed Students t-test).
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8,000
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Figure 3 Mean arterial blood pressure (MAP) after infusion of JT ChAT lymphocytes in wild-type C57Bl/6 mice. (a) Mean arterial blood pressure (MAP)
over ~9 min after infusion of JTChAT lymphocytes or saline. Saline (n = 14); JTChAT cells (n = 713). *P < 0.05, ***P < 0.001, JTChAT cells vs. saline
by ANOVA followed by Bonferroni post hoc test. Error bars, s.e.m. (b) Tracings of MAP over time in mice infused with (1) saline, (2) Jurkat T cells
lymphocytes, or (3) JTChAT lymphocytes, or infusion of JTChAT lymphocytes after with (4) L-NAME or (5) atropine. Arrows indicate the time for infusion
start. The cut in the tracings indicates the period of infusion-related measurement artifacts. (c) MAP change from baseline x time expressed as area
under the curve s.e.m. for mice injected with saline (n = 19), Jurkat T lymphocytes (n = 13), or JTChAT lymphocytes (n = 9). Mice were pre-treated
with L-NAME (n = 7) or atropine (n = 8) as indicated. *P < 0.05, ***P < 0.001 (ANOVA with Bonferroni post hoc analysis). AUC, area under the curve.
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of mice with a genetic disruption of eNOS16 (Supplementary Fig. 18),
as expected from the prior experiments indicating that JTChAT cells
enhance endothelial NO production. Together, these results indicate
that lymphocyte-derived acetylcholine can decrease blood pressure
through a mechanism of endothelium-dependent vasorelaxation.
The identification here of lymphocytes that contribute to blood
pressure regulation reveals a previously unknown mechanism in
cardiovascular homeostasis. The observation that genetic ablation
of ChAT in CD4+ cells results in increased blood pressure in mice
indicates that these cells play a homeostatic role in vasorelaxation and
regulation of blood pressure. T cells have previously been linked to
development of chronic vascular inflammation, atherosclerosis, and
chronic increase in blood pressure1719. Putative pathogenic mechanisms of T cells in hypertension, which are incompletely understood,
include pro-inflammatory cytokine production, T-cell infiltration
into vascular walls, and structural changes and stiffening of arterial
walls secondary to inflammation19,20. It has also been argued that
specific T-cell subtypes may play distinct roles in hypertension. For
example, regulatory T cells may attenuate hypertension by differentially modulating vascular inflammation21. Furthermore, it has been
suggested that neural signaling originating in the brain stem and circumventricular organs may modulate the role of T lymphocytes in
the development of experimental hypertension, and that T cells may
play a role in the cerebral regulation of blood pressure22. The present
findings offer an additional possibility, that a deficiency of CD4 TChAT
cells could contribute to an increase in blood pressure.
We and others have previously implicated CD4 TChAT cells in neural control of inflammation1,23,24. Blood-borne CD4 TChAT cells may
deliver acetylcholine to cells devoid of direct cholinergic innervation,
including vascular endothelial cells, which respond with NO-dependent
vasodilation. Because of the physical constraints within 20- to 40-m
sized arterioles25, blood-borne CD4 TChAT (which are on the order
of ~10 m in diameter) are likely capable of interacting directly with
arteriolar endothelial cells, providing a cellular mechanism of acetylcholine-mediated arteriolar relaxation. Prior unexplained evidence
of decreased ChAT expression in lymphocytes of hypertensive rats as
compared to normotensive rats26 support a role of blood-borne acetylcholine-producing lymphocytes in vasorelaxation. Our findings that
CD4 ChAT mice have an increase in blood pressure render plausible
consideration of whether dysregulation of CD4 TChAT cells and acetylcholine contributes to the pathogenesis of essential hypertension, and
whether this mechanism may underlie hypertension in some cases. This
improved understanding of lymphocyte-derived acetylcholine to mediate endothelial-dependent arterial relaxation suggests that it may be
possible to enhance cellular acetylcholine release in the vasculature to
therapeutically regulate local flow and blood pressure, insights that may
have implications for developing a novel therapy for hypertension.
Moreover, CD4 TChAT cells are regulated by neural signals traveling
in the vagus and splenic nerves1. Ongoing studies of these mechanisms using implanted electrical nerve stimulators27,28 may offer
another strategy to control the activity of blood pressure-regulating
ChAT+ lymphocytes and prevent or reverse hypertension.
Methods
Methods, including statements of data availability and any associated
accession codes and references, are available in the online version of
the paper.
Accession codes. The microarray data discussed in this publication
have been deposited in NCBIs Gene Expression Omnibus29 and are
accessible through GEO Series accession number GSE85915.
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Note: Any Supplementary Information and Source Data files are available in the
online version of the paper.
Acknowledgments
We thank the flow cytometry facility at the Feinstein Institute, the laboratory of
C. Benoist at Harvard Medical School for assistance, and G.K. Hansson for
comments. We thank C. James Kirkpatrick (Institute of Pathology, JohannesGutenberg University, Mainz, Germany), who kindly provided PMEC-ST1.6R
cells. This work benefitted from the data assembled by the ImmGen consortium.
This work was supported by the following grants: NIH, NIGMS RO1 GM57226 to
K.J.T., NIGMS RO1 GM098446 to H.Y. and NIGMS RO1GM089807 to K.J.T. and
V.A.P., VR 2013-3003 to A.A., NIAID R01 029690 to T.L.R., Canadian Institutes
for Health Research FRN 119339 to P.H.B., and Knut and Alice Wallenbergs
Foundation 2014.0212, The Swedish Heart-Lung Foundation 20150767 and Svenska
Lkaresllskapet to P.S.O. Jurkat cells (originally obtained from ATCC) were a gift
from C. Chu, The Feinstein Institute for Medical Research. eNOS-deficient mice were
provided by J. Lundberg and E. Weitzberg, Karolinska Institutet, Stockholm, Sweden.
AUTHOR CONTRIBUTIONS
P.S.O. and K.J.T. conceived the study, planned and performed experiments,
analyzed data and wrote the manuscript. U.A., B.D. and T.W.M. planned
experiments and edited the manuscript. B.E.S., W.M.H. and M.A.C. planned and
performed experiments and analyzed data. M.O. and P.K.G. planned experiments,
analyzed gene expression and edited the manuscript. R.S. and P.H.B. planned
experiments, measured murine blood pressure, performed telemetry and
echocardiography, and analyzed data. M.N.A. and E.J.M. planned, performed and
analyzed experiments with endothelial cells in vitro and murine blood pressure
in response to cell infusion. F.S. and A.A. planned, performed and analyzed
experiments on murine blood pressure in response to cell infusion. A.I., V.A.P.,
K.B., H.Y., S.S.C., S.F.L., C.L., T.L.R. and N.E.H. performed experiments and
analyzed data.
COMPETING FINANCIAL INTERESTS
The authors declare no competing financial interests.
Reprints and permissions information is available online at http://www.nature.com/
reprints/index.html.
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cell data were then averaged for each experiment. To quantify the treatment
response, the average fluorescence in the 1-min intervals immediately before
and after treatment was calculated.
Blood pressure measurement. Arterial catheterization. Adult C57Bl/6 male
mice (2330 g) were anesthetized using isoflurane and maintained on a heating
pad at rectal temperature 37.538 C throughout the experiment. An incision
on the left side of the neck followed by blunt dissection was performed to gain
access to the carotid artery. The artery was tied off toward the head before cannulation. A PE 10 catheter (0.28 mm inner diameter and 0.61 mm outer diameter) was inserted in the artery and secured with USP 6.0 silk suture. Arterial
blood pressure was then monitored through the saline-filled catheter with
recording system (AD-instruments). Cells for infusion were spun down and
resuspended in NaCl before experiments. The suspensions (200 L) were prewarmed and administered via a T-branch on the catheter. In all experiments,
the animals were allowed to stabilize for 5 min after catheter insertion
and after that an initial record of blood pressure with saline injection was
recorded followed by an initial 10-s to 3-min baseline recording. Animals
with hypotension were excluded. The infusion was administered subsequently during approximately 10 s, and blood pressure recorded every 10 s
for 9 min. Blood pressure change over time was assessed by calculating the
area under the curve for the baseline normalized MAP tracing (as exemplified by the shaded areas in Fig. 3b between the 240th and the 780th second) and/or analyzed using 2-way ANOVA followed by Bonferroni post hoc
test using GraphPad Prism. The baseline was calculated as the mean MAP
over 3 min before injection. Jurkat T (JT) or JTChAT lymphocytes (50 107
cells in 200 L saline or the number of cells indicated in the figures) were
administered, with or without pretreatment with atropine (0.05 mg/kg) or LNAME (10 mg/kg). The cells were injected 30 and 10 min after atropine and LNAME, respectively. In experiments with eNOS-deficient mice, ~1 year of age,
saline, Jurkat T cells, or JTChAT cells were infused sequentially after a 10-s base
line recording and blood pressure monitored for ~4 min after each infusion.
Animals with recordings interrupted before 4 min were excluded. In anesthetized male CD4-Cre+/0 ChATflox/flox mice and their littermate controls, a 1.2 F
pressure catheter (Transonic, US) in the right carotid artery was used for
measurements and data acquired using Biopack 3.8 software.
Hemodynamic recording by telemetry. 12-week-old male mice were anesthetized using 2% isoflurane oxygen mixture and placed on a heating pad.
A midline incision was made on the neck and the right common carotid was
isolated. The flexible tip of a hemodynamic telemetry unit (Data Sciences
International, PA-C10) was introduced into the carotid after puncturing the
carotid with a 26-gauge needle. The catheter was secured in place using a 6-0
silk suture. The body of the telemetry unit was placed sub-dermally close
to the abdomen. After 5 days of recovery, arterial pressures were recorded
for 10 s every 1 min over a 48 h period. Data analysis and acquisition were
performed using DataQuest A.R.T software (Data Sciences International).
The investigator was blinded for animal group at time of unit implantation
and for the analysis.
Echocardiography. Male mice were anesthetized using 1.5% isoflurane oxygen mixture. Mice were placed on a heating pad and body temperature was
maintained between 37.3 and 37.6 C for the duration of the measurements. The
investigator was fully blinded for animal group for examination and analysis.
Transthoracic M-mode echocardiographic examination was performed from
the long axis view of the heart at the level of the posteromedial papillary
muscle using a Vevo 7700 system (VisualSonics) with a 30-MHz ultrasonic
linear transducer scanning head. The papillary muscles were not included in
the measurements. Measurements of left ventricle diastolic diameter (LVDd)
and left ventricle systolic diameter (LVDs) were done under Time Motionmode (TM-Mode) and were averaged from at least three cardiac cycles. End
Systolic volume (ESV) and end diastolic volumes (EDV) were calculated
using Vevo770 heart analysis package (volume estimate based on Teichholzs
formula). SV was calculated as EDV-ESV; cardiac output (CO) as SV heart
rate (HR); ejection fraction (EF) as (EDV-ESV)/EDV 100; and fraction
shortening (FS) was calculated as (LVDd-LVDs)/LVDd 100.
Non-invasive blood pressure measurement. Non-invasive blood pressure
measurements were conducted in male CD4-Cre+/0 ChATflox/flox mice and
doi:10.1038/nbt.3663
their littermate controls at 7 weeks of age using the CODA NIBP tail-cuff
device (Kent Scientific Corp., Torrington, CT, USA) as per the manufacturers
instructions. Mice were anesthetized using isoflurane (45% induction, 1.5%
maintenance) and the NIBP and heart rate recorded every 45 s for 1012 min.
The serial measurements were then averaged. All measurements were
conducted during the animals light cycle.
Statistical analysis. Gene expression. RNA isolation and Affymetrix Mouse ST
1.0 gene array hybridization was performed by ImmGen (http://www.immgen.
org/Protocols). Samples with high and comparable array expression levels of
CD3, CD4, and CD44 were included in the analysis.
Data analysis was performed using the R programming language. The raw
data sets from ImmGen13 (Release September 2012) were downloaded from
NCBs GEO data repository (GSE15907). After removal of control, test, and
certain variant subsets (Supplementary Table 2) this data set comprises 27
replicates of 198 different immune cell subsets.
The R/Bioconductor package oligo35 was used for data import and normalization (through rma) and the packages pd.mogene.1.0.st.v1 (Carvalho, B.,
pd.mogene.1.0.st.v1: Platform Design Info for Affymetrix MoGene-1_0-st-v1),
mogene10sttranscriptcluster.db (MacDonald, J.W., mogene10sttranscriptcluster.
db: Affymetrix mogene10 annotation data (chip mogene10sttranscriptcluster),
and the getNetAffx function from the oligo package for annotation of transcripts. This annotation process created a set of 476 transcripts that were identical to the 9th decimal, which were removed. See also Ericson J et al. ImmGen
microarray gene expression data: Data Generation and Quality Control pipeline,
available at https://www.immgen.org/Protocols/ImmGen%20QC%20Docume
ntation_ALL-DataGeneration_0612.pdf. We furthermore removed transcripts
without any annotated gene symbol available, and proceeded to analyze the
remaining data set of 24,925 unique transcripts IDs.
Expression data from ChAT-eGFP+ and ChAT-eGFP subsets were merged
with the ImmGen data using a ratio-based method for batch effect removal36.
To accomplish this, overlapping groups between batches were used as reference samples. The ImmGen data set contains CD4+CD44hiCD62Llo memory
T cells from the spleen, labeled T.4Mem44h62l.Sp. According to available data
on ChAT-eGFP+ and ChAT-eGFP T-cell populations1, >90% of this subset
consists of CD4+CD44hiCD62Llo ChAT-eGFP lymphocytes, and we therefore
used the T.4Mem44h62l.Sp and CD4+CD44hiCD62Llo ChAT-eGFP T-cell
subsets to normalize expression data between ImmGen and ChAT_eGFP
reporter samples. Each batch was first normalized separately using rma.
Then, each sample was divided by the mean of the T.4Mem44h62l.Sp or
CD4+CD44hiCD62Llo ChAT-eGFP subsets, respectively. Data were subsequently merged into a ratio matrix for transcript IDs.
Hierarchical clustering analysis was performed using complete linkage clustering with a Euclidean distance measure. Differential analysis in group comparisons was performed using the R/Bioconductor package limma37. The
David Bioinformatics Annotation Tool (http://david.abcc.ncifcrf.gov/)10,11
was used for Gene Ontology (GO) and KEGG pathway enrichment analysis.
The Gene Ontology enrichment analysis and visualization tool (GOrilla) 9,38
was used for further analysis and visualization of GO terms.
In the analysis of transcription factor expression across the data set, the absolute value of the log-transformed expression values of each transcription factor
in 14 splenic T-cell samples was calculated. Since expression data are in ratio
format this presents a symmetry for high and low expression values. The absolute values of the log-transformed expression ratios were then ranked for each
transcription factor across the 14 samples. For transcription factors that were
ranked highest in the ChAT_eGFP+ T-cell sample, pairwise comparisons for
differential expression between the ChAT_eGFP+ T-cell sample and each of the
other 13 splenic T-cell subsets were performed. Transcription factors for which
ChAT_eGFP+ samples were ranked highest and significantly different in each
pairwise comparison were plotted in a heat map (FDR-adjusted P < 0.05 in any
pairwise comparison between ChAT _eGFP+ T and the other T cells samples).
Sample size was chosen depending on available data (ImmGen) and for
experiments according to prior experience with similar experiments in the
involved research laboratories. Differences between experimental groups
were analyzed using one-way ANOVA followed by Dunnetts or Bonferroni
post hoc analysis or Students t test as indicated. Blood pressure data obtained
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using telemetry were analyzed using repeated measures ANOVA. P < 0.05 was
considered significant.
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30. Lee, P.P. et al. A critical role for Dnmt1 and DNA methylation in T cell development,
function, and survival. Immunity 15, 763774 (2001).
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endothelial cell lines. Lab. Invest. 81, 17171727 (2001).
32. Ye, X., Ding, J., Zhou, X., Chen, G. & Liu, S.F. Divergent roles of endothelial NFkappaB in multiple organ injury and bacterial clearance in mouse models of sepsis.
J. Exp. Med. 205, 13031315 (2008).
33. Ahmed, M.N. et al. Extracellular superoxide dismutase overexpression can reverse the
course of hypoxia-induced pulmonary hypertension. Mol. Med. 18, 3846 (2012).
34. Dong, Q.G. et al. A general strategy for isolation of endothelial cells from murine tissues.
Characterization of two endothelial cell lines from the murine lung and subcutaneous
sponge implants. Arterioscler. Thromb. Vasc. Biol. 17, 15991604 (1997).
35. Carvalho, B.S. & Irizarry, R.A. A framework for oligonucleotide microarray
preprocessing. Bioinformatics 26, 23632367 (2010).
36. Luo, J. et al. A comparison of batch effect removal methods for enhancement of
prediction performance using MAQC-II microarray gene expression data.
Pharmacogenomics J. 10, 278291 (2010).
37. Smyth, G.K. in Bioinformatics and Computational Biology Solutions using R and
Bioconductor (eds. Gentleman, R., Carey, V., Dudoit, S., Irizarry, R. & Huber, W.)
397420 (Springer, New York, 2005).
38. Eden, E., Lipson, D., Yogev, S. & Yakhini, Z. Discovering motifs in ranked lists of
DNA sequences. PLoS Comput. Biol. 3, e39 (2007).
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