P8 INCREASING ENDOTHELIAL INSULIN-LIKE GROWTH
版权申诉
120 浏览量
2023-07-10
18:55:41
上传
评论
收藏 77KB PDF 举报
P6 UNCOVERING MYELOID CELL DIVERSITY IN ATHEROSCLEROSIS USING
MASS CYTOMETRY
Jennifer E Cole
1
, Inhye Park
1
, David Ahern
1
, Lea Dib
1
, Christina Kassiteridi
1
, Dina Danso Abeam
1
,
Michael Goddard
1
, Patricia Green
1
, Pasquale Maffia
2,3,4
, Claudia Monaco
1
1
Kennedy Institute of Rheumatology, Nuffield Department of Orthopaedics, Rheumatology and
Musculoskeletal Sciences, University of Oxford, Oxford, UK;
2
Centre for Immunobiology, Institute of
Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of
Glasgow, Glasgow, UK;
3
Institute of Cardiovascular and Medical Sciences, College of Medical, Veterinary
and Life Sciences, University of Glasgow, Glasgow, UK;
4
Department of Pharmacy, University of Naples
Federico II, Naples, Italy.
Aim: Myeloid cells, particularly macrophages, have critical roles at all stages of atherosclerosis
development. It has become evident that there is a complex continuum of macrophage activation
states particularly in situations where there are dynamic environmental cues. Furthermore, over-
lapping marker expression between subsets has hampered full characterisation and delineation of
macrophage populations from dendritic cells in tissue. We used the multi-dimensionality of mass
cytometry to examine myeloid cell subsets in murine and human atherosclerosis.
Methods and Results: Single cell aortic preparations from atherosclerosis-prone apolipoprotein
E-deficient (ApoE
-/-
) mice fed a chow or a high fat (western) diet or control C57BL/6 mice fed a
chow diet were stained with a panel of 35 metal-conjugated antibodies and analysed using
Cytometry by time of flight (CyTOF). In addition to 13 populations of leucocytes (monocytes,
macrophages, type 1 and type 2 conventional dendritic cells (cDC1 and cDC2), plasmacytoid den-
dritic cells (pDC), neutrophils, eosinophils, B cells, CD4
þ
and CD8
þ
T cells, cd T cell, natural
killer (NK) cells and innate lymphoid (ILC) cells) that accounted for around 95% of all live CD45þ
aortic cells, analysis revealed the presence of multiple macrophage subsets. Progression of athero-
sclerosis was associated with an increase in ‘inflammatory’ and decrease in ‘resident’ macrophage
subsets. CyTOF analysis of human carotid endarterectomy samples also revealed the presence of
multiple myeloid cell populations.
Conclusions: We have used CyTOF to comprehensively map immune cell populations in athero-
sclerosis. Furthermore, our study reveals a switch in the composition of myeloid cell populations
during atherosclerosis development.
P7 LOSS OF KIAA1462, A CORONARYAR TERY DISEASE ASSOCIATED GENE,
DECREASES ATHEROSCLEROSIS
Gillian Douglas, Theodosios Kyriakou, Victoria S. Rashbrook, Edward Drydale, Ayman Al Haj Zen,
Lucy Trelfa, Vedanta Mehta, Ellie Tzima, Hugh Watkins and Keith M. Channon
BHF Centre of Research Excellence, Division of Cardiovascular Medicine, John Radcliffe Hospital, University
of Oxford, UK
Introduction: KIAA1462 is the only coding gene in a chromosome 10 genomic locus consistently
associated with Coronary Artery Disease (CAD) in Genome Wide Association Studies. KIAA1462
has no recognizable functional domains and little homology to other protein families, but has been
previously implicated in endothelial cell proliferation and angiogenesis. We used a murine knock
out (KO) model to investigate the role of Kiaa1462 in the development of atherosclerosis.
Methods and Results: Kiaa1462 global KO mice were generated by replacing exon 3 with a
reporter/selection cassette harbouring LacZ. Kiaa1462 KO mice bred normally with offspring born
in the expected Mendelian ratio. X-gal staining of aortas showed Kiaa1462 expression in vascular
smooth muscle cells (VSMC) and endothelial cells. This was confirmed by qRT-PCR with expres-
sion of Kiaa1462 observed in both isolated endothelial cells and VSMC from WT but not KO
mice. Loss of Kiaa1462 did not alter blood pressure or heart rate. Furthermore, no difference in
vascular contractile function, or either endothelial cell dependent or independent dilation in the
aorta was observed between WT and KO mice.
To assess the role of Kiaa1462 on the development of atherosclerosis we crossed Kiaa1462 KO
mice with hyperlipidaemic ApoE
-/-
mice. Heterozygous breeding pairs were used to generate
matched WT, heterozygous and homozygous KO mice. On an ApoE
-/-
background Kiaa1462 KO
mice were no longer born at the expected Mendelian ratio with a significant (50%) decrease in
KO mice alive 21 days after birth. Surviving KO mice were indistinguishable from their littermates
with no difference in body or organ weight observed. Atherosclerosis was assessed after high fat
feeding for 10 weeks (6 to 16wk of age). Loss of Kiaa1462 caused a significant decrease in plaque
burden in the aortic root of both male and female KO mice vs. WT mice, with no difference
observed between WT and heterozygous KO mice. Despite the decrease in plaque size there was
no significant difference in macrophage or collagen content between genotypes.
Conclusions: Our findings support that Kiaa1462 a GWAS gene candidate plays a critical role in
the development of atherosclerosis.
P8 INCREASING ENDOTHELIAL INSULIN-LIKE GRO WTH F A CTOR-1
RECEPT OR EXPRESSION REDUCES CIRCULATING LEU KOCYTES AND
PROTECTS AGAINSTATHEROSCLEROSIS
MDrozd
1
, NY Yuldasheva
1
, A Maqbool
1
, H Viswambharan
1
,NTWatt
1
,VPalin
1
, S Galloway
1
,A
Skromna
1
, N Makava
1
, SB Wheatcroft
1
,MTKearney
1
, RM Cubbon
1
1
Leeds Institute for Cardiovascular and Metabolic Medicine, LIGHT Laboratories, University of Leeds, UK
Rationale: Experimental evidence suggests that enhanced insulin-like growth factor-1 (IGF-1) sig-
nalling exerts anti-atherosclerotic effects in the arterial wall, but its role in the endothelium
remains unexplored. We studied the effect of increased endothelial IGF-1 receptor (IGF-1R)
expression on atherogenesis in vivo.
Methods and results: We generated ApoE
-/-
mice with transgenic over-expression of human
IGF-1R restricted to the endothelium (hIGFREO-ApoE
-/-
). hIGFREO-ApoE
-/-
mice exhibited a simi-
lar glucose and insulin tolerance to ApoE
-/-
littermates. hIGFREO-ApoE
-/-
mice had significantly
reduced atherosclerosis in the thoracic aorta, aortic arch and aortic sinus, compared to ApoE
-/-
after 12 weeks of western diet. Compared to ApoE
-/-
littermates, hIGFREO-ApoE
-/-
had similar
endothelial-dependent vasomotion and similar expression of serine phosphorylated eNOS and
Akt in whole aorta and endothelial cells (EC). There was a significant reduction in total circulating
leukocytes, and subsets including total myeloid cells, neutrophils and CD11b
þ
Ly6G
-
Ly6C
hi
‘inflam-
matory’ monocytes in hIGFREO-ApoE
-/-
. However, the bone marrow abundance of these leuko-
cyte populations, and Lin
Sca-1
þ
c-Kit
þ
haematopoietic stem cells, was similar. Transplantation of
hIGFREO-ApoE
-/-
bone marrow into irradiated ApoE
-/-
hosts did not alter circulating leukocyte
abundance or atherosclerosis development. hIGFREO-ApoE
-/-
EC and aorta had decreased phos-
phorylation of VE-Cadherin at Y731, a putative regulatory site during leukocyte diapedesis. Bone
marrow vascularity was unaltered in hIGFREO-ApoE
-/-
, although en face analysis of femoral artery
VE-Cadherin junctions demonstrated they were more uniformly organised than ApoE
-/-
litter-
mates. Moreover, hIGFREO-ApoE
-/-
exhibited reduced Evans blue vascular leakage in vivo.
Conclusions: Increased endothelial expression of IGF-1R reduces vascular permeability, leukocy-
tosis and atherogenesis, possibly via VE-Cadherin junction stabilisation.
P9 CYCLIC-AMP DO WN REGULATES EPAC TRANSCRIPTION IN CARDIAC
FIBROBLASTS VIA INHIBITION OF YAP-TEAD ACTIVITY. A NOVEL NEGATIVE
FEEDBACK LOOP CONTROLLING CAMP SIGNALLING
Reza Ebrahimighaei, Andrew Newby and Mark Bond
Department of Translational Health Sciences, University of Bristol, Bristol, BS2 8HW
Exchange Protein Activated by cAMP-1 (EPAC1) is implicated in numerous physiological processes, including
cell proliferation, migration, adhesion and inflammation. Changes in EPAC expression are implicated in several
diseases including cardiac fibrosis and hypertrophy. Elevated cAMP increases EPAC activity but little is known
about how EPAC levels are regulated. Here we investigate regulation of EPAC expression by cAMP.
Forskolin, db-cAMP or adenosine A2B-receptor agonist (BAY 60-6583) significantly reduced EPAC
mRNA levels in cardiac fibroblasts. PKA and EPAC selective agonists acted additively to repress
EPAC mRNA levels. Pre-splice EPAC RNA and activity of an EPAC-promoter reporter vector
was similarly reduced in response to all of these stimuli, suggesting transcriptional repression of
EPAC by cAMP. Phalloidin staining of F-actin demonstrated a rapid disruption of actin polymerisa-
tion in response to elevated cAMP. Furthermore, inhibition of actin polymerisation with
latrunculin-B, cytochalasin-D or Y-27632 (ROCK inhibitor) significantly reduced EPAC mRNA.
Analysis of the EPAC promoter identified a consensus TEAD binding element. Stimulation with
Forskolin or Latrunculin B significantly inhibited TEAD-luciferase activity. Over expression of the
TEAD co-factor YAP, completely reversed forskolin mediated inhibition of TEAD-LUC and EPAC
promoter activity.
Taken together, these data demonstrate that cAMP reduces EPAC gene transcription. Our data
demonstrates that this is mediated, at least in part, by cAMP mediated disruption of the actin
cytoskeleton and inhibition of YAP-TEAD activity. These mechanisms represent a novel negative
feedback loop controlling cAMP signalling, dysregulation of which may contribute towards the
altered EPAC levels that have been observed during disease.
P10 CELL SURF ACE INTERLEUKIN-1a, WHICH DRIVES THE SENESCENCE-
ASSOCIATED SECRET ORY PHENO TYPE (SASP), IS TETHERED VIA IL-1R2 OR
GPI-ANCHORED
J. N. E. Chan, M. Humphry, K. A. Wiggins, L. C. Burzynski, M. C. Clarke
Division of Cardiovascular Medicine, Department of Medicine, Addenbrooke’s Centre for Clinical
Investigation, University of Cambridge, UK
Atherosclerosis is driven by inflammation but the processes that cause chronic inflammation are
not well understood. Macrophages, endothelial cells and vascular smooth muscle cells all undergo
senescence in advanced plaques, resulting in the establishment of the SASP. The SASP is driven by
cell surface IL-1a (csIL-1a) that drives perpetual secretion of inflammatory cytokines that potenti-
ate plaque growth. However, how IL-1a, which lacks a signal peptide or a transmembrane domain,
is tethered to the cell surface is unknown.
Using flow cytometry, we show that murine macrophages express csIL-1a, with expression signifi-
cantly upregulated upon LPS treatment. However, significant false-positive staining can occur with-
out the use of a robust dead cell gate. Furthermore, csIL-1a levels is significantly lower in IL-1R2
-/-
macrophages compared to wild-type, indicating that csIL-1a may be tethered to its cognate recep-
tor IL-1R2. We also find that csIL-1a is in its precursor form and can be cleaved off by thrombin
and calpain. In addition, csIL-1a can also be tethered via a glycosylphosphatidylinositol (GPI)
anchor
We believe the SASP could be a key driver of the chronic inflammation witnessed in vascular dis-
ease. Therefore, elucidating mechanisms that control SASPs are important to identify new targets
that could specifically target vascular inflammation whilst sparing normal host defence.
P11 DEVELOPING NEW TARGETED MOLEC ULAR CONTRAST AGENTS FOR
IMAGING INFLAMMATION OF VULNERABLE PLAQUES
R. J. Evans
1
, J. Hern
andez-Gil
1
, Z. Mohri
2
,K.Y.Chooi
2,5
, B. Lavin-Plaza
4
, A. Phinikaridou
4
, J. E. Pease
3
,
R. Krams
5
, R. Botnar
4
, N. J. Long
1
1
Department of Chemistry;
2
Department of Bioengineering;
3
NHLI, Imperial College London, Exhibition
Road, London, SW7 2AZ;
4
Department of Biomedical Engineering, The Rayne Institute, London, SE1 7EH;
5
Department of Engineering, QMUL, Mile End Road, London, E1 4NS
Vulnerable plaque rupture is the underlying cause of high cardiovascular disease-related mortality,
with no specific imaging agents currently available to detect it. Magnetic Resonance Imaging (MRI)
is non-ionising and non-invasive, with potential for targeted contrast agents. Superparamagnetic
iron oxide nanoparticles (SPIONs) are a non-toxic, long-circulating alternative to gadolinium che-
lates for MRI contrast. For targeting purposes, CX3CL1 is overexpressed in vulnerable plaque but
not present in stable plaque(Cheng et. al.). The aim of this project is to synthesise a range of nano-
particles targeted to CX3CL1 for vulnerable plaque detection with MRI.
Cardiovascular Research Supplements
Poster Presentations S3
Downloaded from https://academic.oup.com/cardiovascres/article-abstract/114/suppl_2/S3/5091440 by guest on 25 October 2018
资源评论
