Oral Abstract Presentations • Abstracts 1-23 will be presented on Thursday • Abstracts 24-59 will be

310 Pages·2017·6.66 MB·English

by  Michele Appley

Oral Abstract Presentations
• Abstracts 1-23 will be presented on Thursday
• Abstracts 24-59 will be presented on Friday

PVD Moderated eAbstract Poster Session
• Abstracts 124, 300, 303, 313, 318, 337, 487, 496, 631 will be presented

Poster Abstract Presentations
• Abstracts 93 – 265 will be presented on Thursday
• Abstracts 299 – 468 will be presented on Friday
• Abstracts 473 – 643 will be presented on Saturday

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ATVB 2017 Scientific Sessions Oral Abstracts
1
Human Monocyte Diversity in Cardiovascular Disease Revealed by Mass Cytometry
Anouk A Hamers, Graham D Thomas, Cheryl Kim, La Jolla Inst for Allergy and Immunology, San Diego,
CA; Anh T Nguyen, Chantel McSkimming, Angela M Taylor, Coleen A McNamara, Univ of Virginia,
Charlottesville, VA; Catherine C Hedrick, La Jolla Inst for Allergy and Immunology, San Diego, CA
Background: Monocytes are critical to the initiation and development of atherosclerosis. To date, 3
distinct human monocyte subsets have been identified based primarily on their expression of the surface
markers CD14 and CD16. With the emerging knowledge of myeloid-derived suppressor cells and other
myeloid subsets, we hypothesized that monocytes are likely more heterogeneous in composition.
Therefore, we set out to use the high dimensionality of mass cytometry to accurately identify and define
monocyte subsets in blood of healthy humans and their changes in cardiovascular patients. Methods:
Heparinized blood from 12 healthy donors and 15 patients with defined cardiovascular disease (CVD)
based on angiography and gensini score was obtained and analyzed by CyTOF mass cytometry. We
employed the Phenograph algorithm to cluster and identify all healthy monocyte subsets based on their
phenotypes using a 40-marker mass cytometry panel. Results: Phenograph identified a total of 15
monocyte clusters in healthy human blood. By performing hierarchical clustering, we were able to group
these clusters into 6 larger meta-clusters and found that most of these meta-clusters fall within the CD14
classical monocyte population, illustrating significant heterogeneity among this monocyte population. Cell
numbers of one of these monocyte meta-clusters were significantly increased in blood from patients with
CVD. We also identified two subsets of nonclassical monocytes in healthy donors. One of these subsets
showed higher expression of the integrin CD61 and tetraspanin CD9, pointing to a possible role for this
subset in patrolling and platelet activation. Conclusion: Monocytes are highly diverse with the
conventional classical subset showing the most diversity. The numbers and frequencies of some of these
monocyte subsets are changed in CVD. Studies to identify their functions in CVD should provide new
information for the role of monocytes in CVD.
Disclosures: A.A.J. Hamers: None. G.D. Thomas: None. C. Kim: None. A.T. Nguyen: None. C.
McSkimming: None. A.M. Taylor: None. C.A. McNamara: None. C.C. Hedrick: None.

2
Aorta Intima-Resident Macrophages Contribute to Atherosclerotic Lesion Initiation
Jesse W Williams, Brian T Saunders, Ki-Wook Kim, Washington Univ in St. Louis, Saint Louis, MO;
Slava Epelman, Univ of Toronto, Toronto, ON, Canada; Kory Lavine, Bernd H Zinselmeyer, Gwendalyn J
Randolph, Washington Univ in St. Louis, Saint Louis, MO
Atherosclerosis is an underlying cause of cardiovascular disease and a leading cause of mortality
worldwide. Macrophage accumulation in atherosclerotic plaque, their uptake of cholesterol, and
subsequent local death drive disease progression. Lipid-laden plaque macrophages are thought to be
exclusively derived from blood monocyte progenitors that are recruited following endothelial damage
induced by cholesterol exposure. In our current study, we focused on characterization of resident
vascular macrophages that reside in the aortic intima in plaque-prone areas, previously identified as
‘vascular dendritic cells’. Using en face whole-mount confocal microscopy of aortas, we confirm a uniform
resident CD64+ CD11c+ CX3CR1+ MHCII+ macrophage population, which is present in C57/BL6 mice
resistant to atherosclerosis. Importantly, they do not express dendritic cell restricted genes zBTB46 or L-
myc. We find aortic macrophages require M-CSF and Flt3 signaling for survival, but are independent of
CCR2, CCR7, and GM-CSF receptor signaling, making them a distinct myeloid population. Lineage-
tracing and parabiosis approaches suggest these cells derive from definitive hematopoiesis and are then
self-maintained independent of blood-progenitors. Using these characterization data, we developed a
labeling strategy to identify resident from recruited macrophages during kinetic studies of lesion
progression. We find that resident aortic macrophages are the first cells to take up lipid following high fat
diet exposure and expand within the arterial wall to form the initial lesion bed. In the absence of resident
macrophages early lipid deposition in the aortic arch is ablated. Finally, utilizing an intravital carotid artery
imaging approach, we identify resident aortic macrophages to be potential mediators of monocyte
recruitment through direct interactions with rolling monocytes on the endothelial surface under diseased
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and steady-states. Overall, these results shift our understanding of the cellular mechanisms responsible
for plaque construction and maintenance.
Disclosures: J.W. Williams: None. B.T. Saunders: None. K. Kim: None. S. Epelman: None. K. Lavine:
None. B.H. Zinselmeyer: None. G.J. Randolph: None.
This Research has received full or partial funding support from the American Heart Association.

3
Cellular Contributions of the Circadian Clock in Atherogenesis
Xueling Li, Ling Ruan, Austin Bentley, Stephen Haigh, Yuqing Huo, Bhupesh Singla, Gabor Csanyi, David
Stepp, David Fulton, R. Daniel Rudic, Medical Coll of Georgia of Augusta Univ, Augusta, GA
Atherosclerosis is a leading cause of death despite the improvements in lipid and blood pressure control.
The circadian clock, a molecular network of genes and proteins that controls 24-hour timing, has emerged
to have a surprising role in the control of metabolic and vascular function. Herein we examined the impact
of circadian rhythm dysfunction in atherogenesis by implementation of vascular transplant and PCSK9
based approaches to induce accelerated lesion development in mice. We find that atherogenesis is
exacerbated in Bmal1-KO aortic grafts immersed in the hypercholesterolemic milieu of ApoE-/- mice. To
assess if atherosclerosis was ‘circadian rhythm dependent’ we subjected wild-type mice to a shortened
light cycle (4L/4D) and induced atherosclerosis by intravenous injection of a human PCSK-9 adeno
associated virus. Atherosclerosis in the jet-lagged PCSK-9 mice was robustly increased relative to the
atherosclerosis observed in WT mice on a normal light cycle (12L/12D), providing further evidence that
circadian rhythm and the circadian clock contribute to atherosclerosis. However, atherosclerosis is a
complex disease that is the net result of interplay between intrinsic (vascular cells) and extrinsic
mechanisms (metabolism, blood pressure, and hormones) and the importance of clock function in
individual cell types is poorly understood. We found that deletion or silencing of key circadian transcription
factors resulted in an enhanced inflammatory and pro-oxidant phenotype with diminished NO production
and greater lipid uptake in both macrophages and endothelial cells. Loss of circadian function in smooth
muscle cells similarly resulted in enhanced production of reactive oxygen species and greater cell
proliferation. Surprising, the silencing of Bmal2 in endothelial cells resulted in greater lipid uptake in
oxLDL treated HAEC as well as increased expression of markers of autophagy, suggesting that Bmal2
may orchestrate numerous output functions in different cell types. In conclusion, we find that the circadian
clock and circadian rhythm have a profound impact on atherosclerosis, to influence vascular cell
inflammatory and lipid uptake responses, and identify an unexpectedly prominent role for the side-partner
of Bmal1, Bmal2.
Disclosures: X. Li: None. L. Ruan: None. A. Bentley: None. S. Haigh: None. Y. Huo: None. B. Singla:
None. G. Csanyi: None. D. Stepp: None. D. Fulton: None. R. Rudic: None.
This Research has received full or partial funding support from the American Heart Association.

4
Deletion of Macrophage Low-density Lipoprotein Receptor-related Protein 1 (LRP1) Accelerates
Atherosclerosis Regression by Increasing CCR7-dependent Egress of Plaque Macrophages
Paul Mueller, Oregon Health and Science Univ, Portland, OR; Lin Zhu, Vanderbilt Univ Medical Ctr,
Nashville, TN; Illaria Giunzioni, Hagai Tavori, Oregon Health and Science Univ, Portland, OR; John M
Stafford, MacRae Linton, Vanderbilt Univ Medical Ctr, Nashville, TN; Sergio Fazio, Oregon Health and
Science Univ, Portland, OR
We previously showed that mice lacking macrophage LDL receptor-related protein 1 (LRP1) undergo
accelerated lesion formation due to increased apoptosis, decreased efferocytosis, and enhanced
macrophage transformation into the inflammatory M1 phenotype. In vitro, LRP1-deficient macrophages
(MΦLRP1-/-) show enhanced plasticity with exaggerated polarization towards either the inflammatory M1-
or the anti-inflammatory M2-phenotype depending on the stimulant (LPS or IL-4, respectively). During
atherosclerosis regression, the M2:M1 macrophage ratio increases as lesion M1 macrophages egress
and inflammation resolves. Thus, we hypothesize that atherosclerosis regression is accelerated in
MΦLRP1-/- mice via enhanced macrophage M2 polarization and CCR7-dependent M1 macrophage
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egress. ApoE-/- mice on high fat diet for 12 weeks were reconstituted with bone marrow from wildtype
(WT) or MΦLRP1-/- mice and then placed on chow diet for 8 weeks. In this model, apoE is reintroduced
into circulation to correct the hyperlipidemia and induce regression of atherosclerotic lesions. A cohort of
apoE-/- mice reconstituted with apoE-/- bone marrow served as baseline controls. Lesions in both WT and
MΦLRP1-/- mice regressed relative to controls (11% and 22%, respectively; p<0.05), but MΦLRP1-/-
lesions were 13% smaller than those of WT mice (p<0.05). LRP1 deletion increased M2 transformation of
macrophages and a higher M2:M1 macrophage ratio (p<0.01) in the plaque. MΦLRP1-/- lesions contained
36% fewer M1 macrophages compared to WT (p<0.01). In vivo studies of reverse cholesterol transport
(RCT) revealed that MΦLRP1-/- have a 1.4-fold higher RCT compared to WT mice (p<0.01). MΦLRP1-/-
lesions contained 2.5-fold more CCR7+ macrophages relative to WT lesions (p<0.01), and in our in vivo
egress assay 4.6-fold more CCR7+ macrophages were found in mediastinal lymph nodes. In vitro, M1-
differentiated MΦLRP1-/- macrophages expressed 1.6-fold higher Ccr7 mRNA compared to WT controls
(p<0.01). Thus, the absence of macrophage LRP1 accelerates atherosclerosis regression due to
enhanced transformation of macrophages into an anti-inflammatory M2 phenotype, increased cholesterol
efflux, and increased CCR7-driven egress of M1 macrophages from lesions.
Disclosures: P. Mueller: None. L. Zhu: None. I. Giunzioni: None. H. Tavori: None. J.M. Stafford:
None. M. Linton: None. S. Fazio: None.

5
The Contribution of Microbiome Alterations to Atherosclerotic Plaque Regression in Mice
Michael Garshick, Tessa Barrett, Felix Zhou, Martin Blaser, Edward Fisher, New York Univ Medical Ctr,
New York CIty, NY
Objective: The human microbiome represents an underexplored driver of atherosclerosis. Alterations to
the intestinal microbiome are associated with, systemic inflammation and upregulation of M1
macrophages (Mφ). Our study aims to determine if alterations to the intestinal microbiome by antibiotic
treatment interferes with atherosclerotic plaque regression in mice.
Methods: ApoE-/- mice were fed a western diet for 16 weeks to develop complex atherosclerosis in aortic
arches. These arches were transplanted into the abdominal aorta of wild-type (WT) mice to model clinical
aggressive lipid management and promote plaque regression. To assess the contribution of the
microbiome to plaque regression, aortic arches were transplanted in WT mice (n=4), and WT mice pulsed
with tylosin (antibiotic, n = 4) 3 days pre-transplant and the duration of the experiment (5 days). Arches
were also transplanted into ApoE-/- mice as a plaque progression control (n =4).
Results: Antibiotic treatment of WT mice with tylosin compared to control WT mice did not change
circulating total cholesterol (TC, 57 ± 21 mg/dl vs. 46 ± 24 mg/dl, p = 0.5), or HDL-C (42 ± 8 mg/dl vs. 34
± 11 mg/dl, p = 0.32). No statistically significant difference in plaque size (117,801 ± 70,921 µm2 vs.
133,286 ± 19,871 µm2 vs. 132,976 ± 40,347 µm2, p = 0.88) or the absolute Mφ content (44,672 ± 27,154
µm2 vs. 32,546 ± 21,226 µm2 vs. 54,154 ± 22,418 µm2, p = 0.47) was observed in WT mice which
received tylosin, compared to control WT and ApoE-/- mice, respectively. The composition of the plaques
however did change: When compared to the control ApoE-/- mice, WT recipient mice had a 26% reduction
in the percent of the plaques occupied by Mφ, while WT mice receiving tylosin had only a 4% reduction (p
= 0.07). There was a 20% difference between the percent of plaque Mφ content in the WT receiving
tylosin, compared to WT controls (38% ± 4.3 vs. 30% ± 6.2, p = 0.07).
Conclusion: Tylosin-treated WT mice undergoing regression had Mφ enrichment in their plaques
compared to WT controls. This study suggests that microbiome alterations can negatively influence
plaque regression. Intestinal microbiome analysis and further studies are needed to confirm and extend
this finding.

Disclosures: M. Garshick: None. T. Barrett: None. F. Zhou: None. M. Blaser: None. E. Fisher: None.

6
A Novel in vivo Endothelial Hierarchy from Progenitor to Mature Endothelial Cells Reveals Key
SoxF-Dependent Differentiation Process
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Jatin Patel, Elke J Seppanen, Mathieu Rodero, Ho Yi Wong, Prudence Donovan, Nicholas M Fisk,
Mathias Francois, Kiarash Khosrotehrani, The Univ of Queensland, Brisbane, Australia
Background: The formation of new blood vessels during adult life is often explained by angiogenesis.
However, an alternate proposal now suggests that neo-vessels form from endothelial progenitors able to
assemble all the intimal layers of vessel structures. Our aim was to define vessel-resident endothelial
progenitors in vivo in a variety of tissues in physiological (aorta, lung) and pathological (wounds, tumors)
situations. Methods and Results: Using common endothelial markers (CD34, CD31, VEGFR2) with flow
cytometry, three sub-populations of endothelial cells could be identified among VE-Cadherin+ and CD45-
cells. These were termed an endovascular progenitor (EVP) harboring CD31lo VEGFR2lo giving rise to
an intermediate CD31intVEGFR2lo transit amplifying (TA) and a definitive differentiated (D)
CD31hiVEGFR2hi population. Confirmation of these populations was demonstrated via lineage tracing
using Cdh5cre ERt2/Rosa-YFP reporter mice. Importantly, EVP cells arose from vascular resident beds that
could not be transferred by bone marrow transplantation, marking their distinction for
hematopoietic/myeloid origin. Furthermore, EVP displayed progenitor like status with a high proportion of
cells in a quiescent cell cycle phase. Only EVP cells and not TA and D cells had self-renewal capacity as
demonstrated by in vitro colony forming and transplant studies in vivo in MatrigelTM plugs in recipient
mice. Through whole RNA sequencing we demonstrated that EVP cells highly expressed genes related to
progenitor function such as Sox9, Il33, Egfr and Pdfgrα, whereas D cells highly expressed genes related
to differentiated endothelium including Ets1&2, Gata2, Cd31, Vwf and Notch. We also determined the
Sox18 transcription factor as having a significant role in defining the endothelial hierarchy, which we
validated through lineage-tracing using Sox18CreERt2/Rosa-YFP mice. In the absence of functional
SOX18/SOXF, EVP progenitors were still present, but TA and D populations were significantly reduced.
Conclusion: In summary, we have demonstrated the existence of an entirely novel endothelial hierarchy,
from EVP to TA to D. This has been demonstrated by the self-renewal, differentiation and molecular
profiling of an EVP.
Disclosures: J. Patel: Ownership Interest; Modest; Angiostem. E.J. Seppanen: None. M. Rodero:
None. H. Wong: None. P. Donovan: None. N.M. Fisk: None. M. Francois: None. K. Khosrotehrani:
Ownership Interest; Modest; Angiostem.

7
In vivo Transcriptional Profiling of Endothelial Cells Identifies Vascular Bed-Specific Changes
Upon Lps-Induced Endotoxemia
Audrey Cleuren, Martijn van der Ent, Kristina Hunker, Hui Jiang, Andrew Yee, David Siemieniak, Santhi
Ganesh, David Ginsburg, Univ of Michigan, Ann Arbor, MI
Endothelial cells (ECs) form a critical barrier between blood and parenchymal cells and play an important
role in many pathologic conditions, including sepsis. ECs are highly adaptive to their microenvironment
and also act as a critical responder to microbial pathogens. Though ECs are thought to display extensive
heterogeneity, detailed profiling of the in vivo EC gene expression program has been limited by the
challenges of isolating ECs from complex tissues and the phenotypic drift associated with manipulation
and expansion of ECs in vitro. We applied an in vivo system in which a conditional hemagglutinin-epitope
tag is targeted into the mouse ribosomal protein Rpl22 locus and specifically activated in ECs, allowing
immunoisolation of endothelial ribosome-associated mRNA. Both EC-selected and total mRNA from
tissue lysates (brain, heart, kidney, liver and lung) were subjected to RNA sequencing followed by
differential expression analysis to determine EC-enriched transcripts. These analyses were performed
under physiologic conditions as well as in LPS injected mice to study transcriptional changes induced in
ECs following endotoxin exposure. LPS-induced endotoxemia resulted in striking changes in the EC
transcriptome (~800 per tissue), and included transcripts associated with known sepsis related
pathophysiology, including impaired hemostasis, leukocyte recruitment and increased vascular
permeability. Gene ontology analysis of transcriptional changes shared between ECs of different tissues
identified cellular response to LPS among the highest enriched biologic processes (adjusted p-value
5.2E-5), together with immune (2.0E-14) and inflammatory responses (4.4E-12). Novel transcripts not
previously associated with ECs or endotoxemia were also identified, as well as a subset of genes
uniquely expressed in distinct vascular beds. In conclusion, our findings demonstrate remarkable
heterogeneity of the EC transcriptome across multiple vascular beds in vivo. The EC response to
endotoxin challenge is also highly heterogeneous across vascular beds and provides new insight into the
endothelial response to infectious challenges, as well as identifying potentially useful biomarkers for the
onset of sepsis and response to therapy.
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Disclosures: A. Cleuren: None. M. van der Ent: None. K. Hunker: None. H. Jiang: None. A. Yee:
None. D. Siemieniak: None. S. Ganesh: None. D. Ginsburg: None.
This Research has received full or partial funding support from the American Heart Association.

8
Endothelial TFEB Regulates Postischemic Angiogenesis via AMPKalpha Activation
Yanbo Fan, Haocheng Lu, Wenying Liang, Yanhong Guo, Ji Zhang, Tianqing Zhu, Yibai Hao, Univ of
Michigan, Ann Arbor, MI; Minerva T Garcia-Barrio, Cardiovascular Res Inst, Morehouse Sch of Med,
Atlanta, GA; Jifeng Zhang, Y Eugene Chen, Univ of Michigan, Ann Arbor, MI
Rationale: Postischemic angiogenesis is critical to limit the ischemic tissue damage and improve the
blood flow recovery. The regulation and the underlying molecular mechanisms of angiogenesis are not
fully unraveled. Transcription factor-EB (TFEB) is emerging as a master gene for autophagy and
lysosome biogenesis. However, the role of TFEB in the vascular disease is less understood.
Objective: We aim to determine the role of endothelial TFEB in postischemic angiogenesis and
underlying molecular mechanism.
Methods and Results: In a murine hindlimb ischemic model, we demonstrated that TFEB was
upregulated in the ischemic skeletal muscle tissue. Utilizing genetically-engineered endothelial cell (EC)
specific TFEB transgenic mice, we investigated the function of TFEB in postischemic angiogenesis. We
observed improved blood perfusion and increased capillary density in the EC-specific TFEB transgenic
mice compared with the wild-type littermates (n = 8-9 for each group, p < 0.01). Furthermore, we found
that blood flow recovery was attenuated in EC-selective TFEB deficient mice compared with control mice
(n =8-9 for each group, p< 0.01). In aortic ring cultures, we found that TFEB transgene significantly
increased the vessel sprouting. Adenovirus-mediated TFEB overexpression promoted EC tube formation
whereas small interfering RNA (siRNA)-mediated TFEB knockdown suppressed tube formation in ECs.
Mechanistically, TFEB activated calcium/calmodulin-dependent protein kinase kinase-β and AMP-
activated protein kinase (AMPK)-α signaling pathway. Through pharmacological inactivation and siRNA-
mediated knockdown of AMPKα, we demonstrated that AMPKα is necessary for TFEB to regulate tube
formation in ECs.
Conclusions: In summary, our data demonstrate that TFEB is a positive regulator of angiogenesis
through activation of AMPKα signaling, suggesting that TFEB constitutes a novel molecular target for
ischemic vascular disease.
Disclosures: Y. Fan: Research Grant; Significant; AHA SDG award. H. Lu: None. W. Liang: None. Y.
Guo: None. J. Zhang: None. T. Zhu: None. Y. Hao: None. M.T. Garcia-Barrio: None. J. Zhang: None.
Y. Chen: Research Grant; Significant; NIH R01.

9
Smooth Muscle Cell Tgfbr2 Deletion in Mice Causes Aortic Hypercontractility and Impaired
Endothelium-Dependent Relaxation
Jay Zhu, Univ of Washington, Seattle, WA; Fatos I Alp Yildirim, Istanbul Univ, Istanbul, Turkey; Stoyan N
Angelov, Hao Wei, David Dichek, Univ of Washington, Seattle, WA
Background: Abnormal smooth muscle cell (SMC) TGF-β signaling is proposed as a critical driver in the
development of thoracic aortic aneurysms and dissections (TAAD) associated with Marfan and Loeys-
Dietz Syndromes as well as nonsyndromic TAAD. However, the mechanisms by which altered SMC TGF-
β signaling causes TAAD are poorly understood. Others have proposed that loss of SMC TGF-β signaling
causes TAAD by impairing SMC contractility, leading to aortic medial degeneration and dilation. However,
mice generated in our lab with deficient SMC TGF-β signaling (due to SMC-specific deletion of the type II
TGF-β receptor) have thicker aortic medias and increased mRNA encoding SMC contractile proteins.
These observations predict increased contractility.
Methods & Results: We addressed this apparent contradiction experimentally by measuring vasomotor
function (by tension myography) and contractile protein expression (by immunoblotting) in aortas of mice
with normal or deficient SMC TGF-β signaling. Isolated aortic rings from mice with deficient SMC TGF-β
signaling showed increased contraction to phenylephrine (Emax: 13.5 mN vs 7.8 mN in controls;
p<0.0001; n=19-20) and potassium chloride (Emax: 7.5 mN vs 5.7 mN in controls; p<0.0001; n=19-20).
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Moreover, levels of smooth muscle myosin heavy chain protein were at least as high in aortas with
deficient SMC TGF-β signaling as in control aortas, consistent with their capacity to generate increased
contractile force. Surprisingly, aortic segments from mice with deficient SMC TGF-β signaling also had
impaired endothelium-dependent relaxation to acetylcholine (Emax: 37% vs 97% in controls; p<0.0001;
n=19-20). Endothelium-independent relaxation to sodium nitroprusside was similar between the two
groups. CD31 immunostaining of vessel segments revealed equivalent endothelial integrity in both
groups.
Conclusion: Physiologic SMC TGF-β signaling is an important determinant of both SMC contractility and
endothelial function. Disruption of physiologic SMC TGF-β signaling may lead to TAAD through direct
effects on SMC as well as through indirect effects on endothelial function. Our results also suggest an
unanticipated role for SMC TGF-β signaling in regulating endothelial-mediated vasomotor function.
Disclosures: J. Zhu: None. F.I. Alp Yildirim: None. S.N. Angelov: None. H. Wei: None. D. Dichek:
None.

10
Endothelial FGF Protects Against Pulmonary Hypertension
Kel Vin Woo, Carla Weinheimer, Attila Kovacs, David Ornitz, Washington Univ, Saint Louis, MO
Pulmonary hypertension (PH) is a debilitating disease where 1 in 4 patients will die within five years of
diagnosis. Pathologic changes of endothelial cell function, together with smooth muscle and adventitial
hyperplasia increase pulmonary vascular resistance. Consequent elevation in right ventricular pressure
ultimately causes right heart failure.
Expression of Fibroblast Growth Factor Receptors (FGFRs), FGFR1 and FGFR2, are elevated in lung
samples from PH patients; however, the impact of these receptors on endothelial cell function, and
endothelial to smooth muscle interaction is poorly understood.
We hypothesize that activation of endothelial FGFR1 and FGFR2 promotes endothelial cell survival,
elaborating signals that protect against pulmonary hypertension via inhibition of smooth muscle cell
recruitment.
We used the Tie2-Cre transgene to conditionally inactivate Fgfr1 and Fgfr2 in endothelial cells.
Experimental mice with genotype Tie2-Cre; Fgfr1f/f; Fgfr2f/f (DCKO) and control Fgfr1f/f; Fgfr2f/f (DFF) were
challenged with 10% hypoxia for 2 weeks. At the end, right ventricular pressure (RVp) was measured by
cardiac catheterization. Compared to mice in normoxia, control littermates in hypoxia demonstrated
significant increases in RVp, and RV to left ventricle + septum (LV+S) weight ratio, consistent with
development of PH. DCKO mice demonstrate further elevation in RVp and an increase in the RV to LV+S
weight ratio, demonstrating worsening PH. We also observed formation of plexiform lesions in DCKO
mice, suggestive of a severe pathology. We found a previously unreported involvement of FGF10 in
pulmonary hypertension. FGF10 expression was decreased in hypoxia-challenged DCKO mice as
compared to both DCKO littermates on room air, and to hypoxia challenged DFF control mice.
Our data suggests that endothelial FGFR1 and FGFR2 activation may protect against pulmonary
hypertension. Further studies are underway to elucidate the role of FGF10 and its mechanism in the
pathology. We will also further identify associated FGF ligands, associated signaling mechanisms, and
endothelial to smooth muscle interactions.
Disclosures: K. Woo: None. C. Weinheimer: None. A. Kovacs: None. D. Ornitz: None.

11
Effects of Canakinumab in Patients with Peripheral Artery Disease
Kerry S Russell, Denise Yates, Andrea Feller, Tianke Wang, Ping Chen, Timothy Clough, Laurence
Colin, Novartis Insts for BioMedical Res, Cambridge, MA; Lucy LaPerna, Remington Davis, Columbus,
OH; Mustafa M Shennak, Triumpharma, Amman, Jordan; Holger Lawall, Akademie fuer
Gefaesskrankheiten, Hamburg, Germany; Sigrid Nikol, Asklepios Klinik St Georg, Hamburg, Germany;
William Smith, NOCCR, Knoxville, TN; Thomas Forst, Profil Mainz, Mainz, Germany; Oliver J Mueller,
Univ of Heidelberg, Heidelberg, Germany; John Hoekstra, Natl Clinical Res, Richmond, VA; Marek Piatek,
Aspire Clinical Res, Glendale, AZ; Elizabeth V Ratchford, Johns Hopkins Univ, Baltimore, MD;
Christopher M Kramer, Univ of Virginia Health System, Charlottesville, VA; Craig T Basson, Novartis Insts
for BioMedical Res, Cambridge, MA
7 | Page
Background Peripheral artery disease (PAD) affects 8.5 million people in the US. PAD patients are at
high risk for cardiovascular events, and their quality of life is often significantly impaired by decreased
mobility. Interleukin-1β (IL-1β) may play an important role in this disease by promoting inflammatory
responses that drive atherosclerotic plaque progression and impair vascular function. We sought to test
whether interruption of IL-1β signaling would improve patient mobility and decrease plaque progression in
the lower extremities. Methods 38 patients (mean age 65; 71% male) with symptomatic PAD (confirmed
by ankle-brachial index) were randomized 1:1 to receive Canakinumab (150 mg subcutaneously) or
placebo monthly for up to 12 months. Plaque volume in the superficial femoral artery (SFA) was assessed
serially using 3.0T MRI. Mobility was assessed serially using the 6-minute walk test (maximum and pain-
free walking distance). Results Canakinumab was safe and well-tolerated. 12 patients discontinued (8
placebo, 4 Canakinumab). MRI data (from 31 patients at 3 months; 21 patients at 12 months) showed no
evidence of plaque progression in the SFA at either time point in placebo-treated patients; nor was there
a change in plaque volume in the Canakinumab-treated group. There was a serial and significant
improvement in placebo-adjusted maximum and pain-free walking distance observed as early as 3
months after treatment with Canakinumab (58-meter improvement over placebo in pain-free distance at 3
months, P=0.01). Two placebo-treated patients required peripheral vascular interventions due to
progression of disease; however, no Canakinumab-treated patients required revascularization during the
study. Canakinumab decreased markers of systemic inflammation (IL-6 and hsCRP). Conclusions
Treatment with Canakinumab may improve maximum and pain-free walk distance in patients with
symptomatic PAD. In conjunction with results soon to be reported for the CANTOS trial of Canakinumab
for secondary prevention of cardiovascular events, additional studies may provide support that inhibition
of IL-1β signaling can improve symptoms and function in this patient population with high unmet need.
Disclosures: K.S. Russell: Employment; Significant; Novartis Employee. D. Yates: Employment;
Significant; Novartis Employee. A. Feller: Employment; Significant; Novartis Employee. T. Wang:
Employment; Significant; Novartis Employee. P. Chen: Employment; Significant; Novartis Contracted
Employee. T. Clough: Employment; Significant; Novartis Employee. L. Colin: Employment; Significant;
Novartis Employee. L. LaPerna: Other; Significant; Investigator on Novartis-sponsored Trial. M.M.
Shennak: Other; Significant; Investigator on Novartis-sponsored Trial. H. Lawall: Other; Significant;
Investigator on Novartis-sponsored Trial. S. Nikol: Other; Significant; Investigator on Novartis-sponsored
Trial. W. Smith: Other; Significant; Investigator on Novartis-sponsored Trial. T. Forst: Other; Significant;
Investigator on Novartis-sponsored Trial. O.J. Mueller: Other; Significant; Investigator on Novartis-
sponsored Trial. J. Hoekstra: Other; Significant; Investigator on Novartis-sponsored Trial. M. Piatek:
Other; Significant; Investigator on Novartis-sponsored Trial. E.V. Ratchford: Other; Significant;
Investigator on Novartis-sponsored Trial. C.M. Kramer: Other; Significant; Investigator on Novartis-
sponsored Trial. C.T. Basson: Other; Significant; Novartis Employee.

12
Volume of Calcium in the Descending Thoracic Aorta Predicts All Cause Mortality Beyond
Coronary Artery Calcium: The Multi-Ethnic Study of Atherosclerosis
Isac C Thomas, Matthew A. Allison, Nketi I. Forbang, Univ of California, San Diego, La Jolla, CA; Erin D.
Michos, Johns Hopkins Univ, Baltimore, MD; WT Longstreth Jr., Univ of Washington, Seattle, WA; Nathan
Wong, Univ of California, Irvine, CA; Wendy Post, Johns Hopkins Univ, Baltimore, MD; Michael H. Criqui,
Univ of California, San Diego, La Jolla, CA
Introduction: Coronary artery calcium (CAC) volume and density differentially predict incident
cardiovascular disease (CVD), with CAC density inversely associated with these outcomes. Whether
similar associations exist between descending thoracic aortic calcium (DTAC) volume and density and all
cause mortality (ACM) are unknown. We hypothesized that DTAC volume and density predict ACM
independently of CAC.
Methods: The Multi-Ethnic Study of Atherosclerosis enrolled 6,814 participants free of clinical CVD at
baseline and followed them for incident adverse events. Cardiac CT at baseline visualized the segment of
the descending thoracic aorta posterior to the heart. Only participants with prevalent DTAC were included
(necessary to evaluate DTAC density). DTAC and CAC volumes were natural log transformed to adjust
for skewness. Cox regression models estimated the associations of DTAC volume and density with ACM
after adjustment for age, gender, ethnicity, CVD risk factors, statin use, and CAC volume and density.
The incremental predictive values of DTAC volume and density were evaluated by area under receiver
operating characteristic (AUC) curves.
Results: Of the total cohort, 1,850 participants (27%) had prevalent DTAC and 491 deaths occurred over
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10.3 years. In separate regression models, DTAC volume was independently associated with ACM after
adjustment for CAC volume (HR 1.21 [95% CI 1.09-1.35]) and additional adjustment for CAC density
(1.18 ([1.06-1.32]). After the same adjustments, DTAC density was not significantly associated with ACM
(0.94 [0.84-1.06]). The AUC for the base Model 1 (risk factors + CAC volume) was 0.706 (0.680-0.732),
which increased to 0.716 (0.690-0.742) with the addition of DTAC volume in Model 2 (p=0.03 compared
to Model 1). Further addition of DTAC density in Model 3 did not improve the AUC significantly (0.717
[0.692-0.743], p=0.23 compared to Model 2).
Conclusions: In a cohort free of baseline clinical CVD, DTAC visualized on cardiac CT was common.
When DTAC was present, DTAC volume (but not density) was independently associated with ACM.
DTAC volume also significantly improved ACM risk prediction beyond risk factors and CAC volume.
Disclosures: I.C. Thomas: None. M.A. Allison: None. N.I. Forbang: None. E.D. Michos: None. W.
Longstreth: None. N. Wong: None. W. Post: None. M.H. Criqui: None.

13
Inhibition of Nudt6 Stabilizes Advanced Atherosclerotic Plaques
Hong Jin, Yuhuang Li, Ekaterina Chernogubova, Alexandra Bäcklund, Stina Sellberg, Changyan Sun,
Karolinska Inst, Stockholm, Sweden, Sweden; Albert Busch, Technical Univ Munich, Munich, Germany,
Germany; Suzanne M Eken, Ljubica Perisic, Ulf Hedin, Karolinska Inst, Stockholm, Sweden, Sweden;
Pavlos Tsantilas, Hans-Henning Eckstein, Jaroslav Pelisek, Technical Univ Munich, Munich, Germany,
Germany; Lars Maegdefessel, Karolinska Inst, Stockholm, Sweden, Sweden
Natural antisense transcripts (NATs), a non-coding RNA subclass, being transcribed in antisense
direction to protein coding genes, are an intriguing novel class of targetable modulators, exerting crucial
effects on gene expression. Aim of the current study was to investigate the contribution of NATs to
atherosclerotic plaque vulnerability.
Using laser capture micro-dissection, we isolated fibrous caps tissue of carotid artery plaques from 20
symptomatic patients with ruptured lesions vs. 20 samples from asymptomatic patients with stable
lesions. A human transcriptome array (HTA; GeneChip 2.0) was used to profile the expression of all
currently annotated RNA transcripts. Nucleoside diphosphate-linked moiety X motif 6 (NUDT6) was
identified as one of the most significantly up-regulated transcripts in fibrous caps of ruptured lesions.
Interestingly, NUDT6 is an established antisense RNA targeting the fibroblast growth factor 2 (FGF2). Of
importance, FGF2 was among the most significantly down-regulated transcripts in ruptured lesions,
corresponding to elevated NUDT6 expression.
In situ hybridization in both, human and mouse carotid atherosclerotic plaques, confirmed substantially
higher expression levels of NUDT6 in ruptured lesions compared to stable. In addition, in situ
hybridization revealed a distinct co-localization with smooth muscle cells (SMCs) in advanced plaques.
Overexpression of NUDT6 in cultured human carotid artery SMCs effectively limited FGF2 on the mRNA
as well as protein level. Furthermore, reduction of NUDT6 via siRNA stimulated proliferation and blocked
apoptosis in SMCs. In an inducible atherosclerotic plaque rupture model using incomplete ligation and
cuff placement on common carotid arteries of male apoE-/- mice, NUDT6 inhibition with gapmeRs was
able to significantly improve SMC survival rates, leading to thicker fibrous caps, and to reduce the plaque
rupture rate compared to scramble-gapmeR control-treated mice (22% vs. 63%, p= 0.03).
The present study presents NUDT6 as a novel crucial antisense regulator of fibrous cap stability through
steering SMC survival via targeting its sense RNA transcript FGF2.
Disclosures: H. Jin: None. Y. Li: None. E. Chernogubova: None. A. Bäcklund: None. S. Sellberg:
None. C. Sun: None. A. Busch: None. S.M. Eken: None. L. Perisic: None. U. Hedin: None. P.
Tsantilas: None. H. Eckstein: None. J. Pelisek: None. L. Maegdefessel: None.

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14
Characterization of the Oxidized Phospholipid Modification of Apolipoprotein(a) Kringle KIV10:
Insights into the Site of OxPC Addition
Corey A Scipione, Robarts Res Inst, London, ON, Canada; Travis DeWolfe, James W Gauld, Univ of
Windsor, Windsor, ON, Canada; Michael B Boffa, Western Univ, London, ON, Canada; Amir Ravandi, St.
Boniface Hosp Res Ctr, Winnipeg, MB, Canada; Marlys L Koschinsky, Robarts Res Inst, London, ON,
Canada
Elevated plasma levels of lipoprotein(a) (Lp(a)) are an independent and causal risk factor for coronary
heart disease and aortic valve stenosis. Lp(a) consists of a low density lipoprotein (LDL)-like particle
covalently linked to the unique glycoprotein apolipoprotein(a) (apo(a)). We have shown that apo(a)
contains a covalent oxidized phosphocholine (oxPC) adduct on the KIV10 domain, and that perturbation of
the strong lysine binding site (LBS) in this kringle results in a lack of covalent oxPC addition. We have
implicated this modification in proinflammatory processes, such as the ability of apo(a) to induce
interleukin-8 expression in macrophages. Apo(a) from Old World monkeys and apes lacks covalent oxPC
modification and contains mutations in KIV10, some of which impact the LBS. To identify the amino acids
in human apo(a) that are covalently modified by oxPC, we mutagenized a KIV10KV di-kringle apo(a) that
contains covalent oxPC modification. We mutated to alanine all residues that are either substituted in
primate apo(a) or that can act as acceptors for covalent oxPC addition. The resulting variants were then
subjected to immunoblotting analysis with E06, an IgM antibody that binds to oxPC, and assessment of
lysine-binding ability using affinity chromatography. Mutation of His33 to Ala abolishes oxPC modification
of apo(a) while retaining the lysine binding ability of KIV10. Thus, we have identified the long-sought
location of the oxPC modification of apo(a). Molecular dynamic simulations of the oxPC-deficient mutant
show no gross changes in the structure of KIV10, in contrast to mutations that abolish both lysine binding
ability and oxPC addition that result in a collapsed lysine binding pocket. Using mass spectrometry, we
have also identified noncovalently-associated oxPC present on apo(a). Interestingly, the abundance of
these species appears to be reduced in the apo(a) variant containing a mutation in the strong lysine
binding site in KIV10. Our findings allow the ability to assess the contributions to pathogenesis of the
strong lysine binding site and oxPC modification of apo(a) kringle IV type 10 independently, and thus will
enhance our understanding of the mechanisms by which Lp(a) contributes to atherothrombotic diseases.
Disclosures: C.A. Scipione: None. T. DeWolfe: None. J.W. Gauld: None. M.B. Boffa: None. A.
Ravandi: None. M.L. Koschinsky: Consultant/Advisory Board; Modest; Lipoprotein(a) Foundation.
15
Serum Amyloid A is Not Just an HDL-Associated Lipoprotein
Patricia G Wilson, Joel C Thompson, Preetha Shridas, Frederick C de Beer, Nancy R Webb, Lisa R
Tannock, Univ Of Kentucky, Lexington, KY
Serum Amyloid A (SAA) is traditionally thought to be only found with HDL; however, recently several
groups have found that SAA is found on apoB-containing lipoproteins (LDL and VLDL) under some
circumstances. The goal of this study was to determine the relative lipoprotein association of SAA. Native
human acute phase SAA was isolated from acute phase plasma collected from patients undergoing
cardiovascular bypass surgery; the SAA was purified and delipidated. Plasma was collected from a group
of healthy, non-obese humans with low levels of SAA (< 2 mg/L) and lipoproteins (VLDL, LDL and HDL)
were isolated by density gradient ultracentrifugation. The delipidated SAA (at 2 concentrations) was
incubated in vitro with the various lipoprotein preparations then samples were re-isolated by fast protein
liquid chromatography and SAA was analyzed by ELISA and immunoblot. When SAA was incubated with
any single lipoprotein all of the SAA was found associated with that lipoprotein and none remained in a
lipid-free form. When SAA was incubated with a mixture of VLDL, LDL and HDL (based on equal protein
and corresponding to concentrations in plasma) a majority of SAA (50-60%) was found on HDL with the
remaining SAA found on VLDL and LDL. When SAA first complexed to HDL was added to a mixture of
SAA-free LDL and VLDL the majority of SAA remained with the HDL (76-86%) but 5-10% of the SAA was
found on each of LDL and VLDL. When SAA first complexed to either apoB-containing lipoprotein was
then added to a mixture of HDL and the other apoB-containing lipoprotein most SAA moved to HDL (55-
70%) but the remainder was found on the apoB-containing particles. Thus, SAA can move between
lipoprotein particles in vitro. To determine if the presence of SAA on apoB-containing lipoproteins had a
functional effect we evaluated lipoprotein-proteoglycan binding affinity. Proteoglycan mediated lipoprotein
retention in the vessel wall is thought to be one of the key steps in initiation of atherosclerosis. Compared
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