bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 435: Vascular Gene Therapy with Apolipoprotein A-I in a Rabbit Model of Atherosclerosis Regression
Authors: Wacker Bradley K , Zhang Jingwan , Dronadula Nagadhara , Dichek David A
Published: June 2021

Background:
Gene therapy, delivered directly to the vascular wall, could potentially protect against atherosclerotic lesion growth or regress existing lesions. Previously, we demonstrated that use of a helper-dependent adenoviral vector (HDAd) to express apolipoprotein (apo) A-I in carotid endothelium of fat-fed rabbits reduced carotid atherosclerosis measured 4 wk after vector infusion. Here we tested whether the same HDAd could promote atherosclerosis regression when delivered to existing lesions.

Methods:
To generate atherosclerotic lesions, rabbits were fed a high-fat diet for 4 wk, then underwent bilateral carotid artery surgery with brief intraluminal infusion of DMEM. This protocol yields large lipid- and macrophage-rich lesions within 6 mo. Postoperatively, diets were adjusted to maintain plasma cholesterol of 200 - 800 mg/dl. 6 mo later, rabbits underwent bilateral carotid gene transfer (HDAdApoAI on one side; HDAdNull on the other, with sides randomized) and were changed to chow diet. Carotids were harvested after 3 d (for RNA analysis) and after 7 wk (for analyses of RNA, DNA, and histology). Histologic sections were stained with H&E, oil red O, or antibodies that detect macrophages, smooth muscle actin, ICAM1, and VCAM1.

Results:
ApoA-I mRNA was detected only in arteries transduced with HDAdApoAI and was present in all HDAdApoAI-infused arteries both at 3 d and 7 wk after infusion. When compared to HDAdNull-infused arteries (n = 26), HDAdApoAI-infused arteries (n = 26) had 30 - 40% less median intimal lesion volume, lipid content, and macrophage content but these differences were not statistically significant (P = 0.3 - 0.9). Post-hoc subgroup analysis of small-to-moderate-sized lesions (n = 20 per group; excluding lesions with volume >1 SD above the mean of all lesions) showed that HDAdApoAI-infused lesions had 50 - 70% less median intimal volume (P = 0.04), lipid content (P = 0.02), and macrophage content (P = 0.03).

Conclusions:
7 wk of vascular gene therapy with HDAdApoAI did not consistently cause lesion regression. However, subgroup analysis showed that HDAdApoAI significantly accelerated regression of small-to-moderate-sized atherosclerotic lesions. Prospective testing is required to evaluate the reproducibility of this finding.
Abstract 437: Cd146 Deficiency Leads to Accelerated Atherosclerosis in Mice Through Upregulation of Rantes
Authors: Blin Muriel G , Bachelier Richard , Fernandez Samantha , Guillet Benjamin , Fallague Karim , Robert Stéphane , Heymes Christophe , Bardin Nathalie , Blot-Chabaud Marcel , Dignat-George Francoise , Leroyer Aurélie S
Published: June 2021

Rationale:
The progression of atherosclerosis is based on the continued recruitment of leukocytes in the vessel wall. The previously described role of CD146 in leukocyte infiltration
in vitro
suggests a role for this endothelial junction molecule in the pathogenesis of atherosclerosis. However, its involvement in atherosclerotic plaques formation has never been investigated.

Objective:
We evaluated the role of CD146 in atherogenesis.

Methods and Results:
CD146 -/-/ApoE -/- and ApoE -/- mice were fed a Western diet for 24 weeks and were monitored for aortic wall thickness using high frequency ultrasound. The arterial wall thickness was significantly higher in CD146 deficient mice. We evidenced a significant increase of atheroma in both total aortic lesion and aortic sinus of CD146 deficient mice. In addition, atherosclerotic lesions were more inflammatory since CD146 deficient plaques contained more neutrophils and more macrophages. During atherosclerosis, circulating neutrophils were significantly increased in the absence of CD146. Consistent with the higher recruitment of inflammatory cells to the atheroma, we demonstrated that RANTES was up-regulated in CD146 deficient atherosclerotic arteries. In addition, CD146 deficient mice presented significant higher levels of circulating RANTES during atherosclerosis. Finally, we showed that macrophages were the source of RANTES and its production by CD146 null macrophages was also significantly increased through a mechanism dependent of p38-MAPK signaling pathway.

Conclusions:
Our data indicate that CD146 deficiency is associated with the upregulation of RANTES and increased inflammation of atheroma, which could influence the atherosclerotic plaque fate. Thus, these data identify CD146 as a potential new target for atherosclerosis treatment.
Abstract 441: Gamma-glutamyltransferase and Markers of Subclinical Atherosclerosis in Patients with Psoriasis
Authors: Demircioglu Kenan , Aksu Feyza , Caliskan Mustafa , Yilmaz Yusuf
Published: June 2021

Introduction:
Gamma-glutamyltransferase(GGT) plays a catalytic role in degradation of glutathione. Serum GGT is accepted as a marker of oxidative stress.The aim of this study is to investigate the relationship between serum GGT levels and epicardial adipose tissue (EFT) thickness, carotid intima media thickness (CIMT) measurements in patients with psoriasis.

Methods:
The study population included 89 patients with psoriasis and 79 healthy volunteers. After overnight fasting, blood samples were taken for to determine blood glucose levels and establishing cholesterol profiles including TG, TC, LDL cholesterol and high-density lipoprotein (HDL) cholesterol; GGT; and high- sensitivity C-reaktive protein (hs-CRP) levels. A high-resolution B-mode ultrasound machine (Toshiba, aplio XU) with a 7.5 MHz linear transducer used for examing CIMT.The right common carotid artery (CCA), approximately 1 cm proximal to the bifurcation, was longitudinally selected and CIMT was defined as the distance between the intima and the media.

Results:
89 patients with psoriasis (age:41.7±10.9 years;41 women, 48 men), and 71 healthy control subjects (age:40.4±8.2 years;39 women, 32 men) were included. There were no significant variation for age and sex between two groups(p>0.05).The hs-CRP and GGT values were significantly higher in psoriasis, compared with the controls (hs-CRP:1.35(0.9-3.6)mg/l for psoriasis group, 0.45(0.29-0.79)mg/l for control group, p<0.001; GGT:20.6±9.6 U/l for psoriasis group, 16.7±8.0 U/l for control group, p=0.02. In psoriatic patients, CIMT and EFT were significantly inreased (0.60(0.50-0.68)mm vs. 0.50 (0.40-0.60)mm;p=0.007, 0.67±0.20cm; 0.27±0.12cm; p<0.001, respectively) compared with the control group. CIMT significantly positively correlated with EFT, age, BMI, diastolic BP and GGT.EFT significantly positively correlated with GGT, CIMT, age, hs-CRP, systolic BP and TG and negatively correlated with HDL cholesterol.

Discussion:
The pathophysiology of atherosclerosis in psoriasis is not fully explained.GGT may be used as an indicator of subclinical atherosclerosis like CRP.
Abstract 459: Differential Responses of Bone Marrow-Derived Macrophages to Lipopolysaccharide in the Treml1 Null Mouse
Authors: Pacheco Amanda I , Washington Anthony V , Gonzalez Marieli
Published: June 2021

Atherosclerosis, the hardening and narrowing of the arteries, is a chronic inflammatory disease driven by a crosstalk of signaling molecules between circulating immune cells and the vessel wall. In the classic view of this cardiovascular disease, hyperlipidemia initiates an inflammatory cascade that promotes macrophage infiltration into the vessel wall leading to a feedback mechanism which exacerbates lesion growth and ultimately, atherothrombosis. Our laboratory has characterized a novel atherosclerotic mice model, in which deletion of the platelet receptor Triggering Receptor Expressed on Myeloid Cells (TREM)-Like Transcript-1 (TLT-1) on a apolipoprotein E (

apoe
-/-
/treml1
-/-
)

background dampens early macrophage infiltration into the vessel wall. This leads to decreased lesion size, instability, and calcification of atherosclerotic lesions. We have demonstrated that one of the mechanisms for dampened lesion progression is decreased platelet activation and we hypothesize that the macrophage infiltration differences may also be due to fundamental differences in macrophage phenotypic functions. In order to further define the mechanism by which TLT-1 modulates macrophage infiltration, we analyzed macrophage function
in vitro
. Analysis of bone marrow-derived macrophages (BMDM) from

apoe
-/-
/treml1
-/-

and

apoe
-/-
/treml1
-/+

mice revealed no significant differences in the phagocytic function of long chain fatty acids. However, analysis of basal levels of monocyte chemoattractant protein-1 (MCP1), a key chemokine regulating monocyte infiltration, were significantly decreased in the apoe
-/-
/treml1
-/-
BMDM supernatants. Accordingly, levels of Interleukin 6 (IL6) and Interleukin 10 (IL10) were significantly decreased in the apoe
-/-
/treml1
-/-
BMDM supernatants after two hours of lipopolysaccharide (LPS) stimulation (p<0.05; n=5-8). These findings define an underlying difference in the BMDM secretory phenotype of the apoe
-/-
/treml1
-/-
mice. However, it also opens the possibility of
treml1
expression in macrophages, which may account for these differences. We will now investigate if an alternate isoform of
treml1
is expressed in macrophages or at some period of their differentiation.
Abstract 460:
64
Cu-DOTATATE for in vivo Positron Emission Tomography Imaging of Somatostatin Receptor 2 Expressing Macrophages in a Göttingen Minipig Model of Atherosclerosis
Authors: Pedersen Sune Folke , Ludvigsen Trine Pagh , Vegge Andreas , Schumacher-Petersen Camilla , Ripa Rasmus Sejersten , Pedersen Henrik Duelund , Kjær Andreas
Published: June 2021

Background:
Somatostatin receptor subtype 2 (SSTR
2
) is expressed by activated macrophages which are important effector cells in atherogenesis and a major constituent of atherosclerotic plaques. SSTR
2
can be targeted non-invasively in vivo using the tracer
64
Cu-DOTATATE and positron emission tomography (PET).

Methods:
Male castrated Göttingen minipigs were treated according to two diet regimes for 43 weeks: chow (lean control; n=1) or high-fat/high-cholesterol diet (HFHC, obese group; n=2). Plasma lipids: total cholesterol (TC) and triglycerides (TG) were measured and SSTR
2
expression was assessed using hybrid positron emission tomography/magnetic resonance (PET/MR) imaging of the abdominal aorta, one hour (range: 60-63 minutes) post injection with 200 MBq (range 202-214 MBq) of
64
Cu-DOTATATE. Consectutive regions of interest (ROIs) were drawn guided by MR to include vessel wall and lumen of the abdominal aorta to calculate standardized uptake values (SUVs). Target-to-background (TBR) ratios were calculated using right atrium SUVs for blood pool correction (SUV
vessel
/SUV
right atrium
= TBR) and reported as mean and maximal values (TBR
mean
; TBR
max
).

Results:
Three minipigs (age 17 months) were included and PET/MR was completed in all animals. Mean uptake of
64
Cu-DOTATATE was lowest for the abdominal aorta in the lean control: TBR
mean
= 0.44 and TBR
max
= 0.73 (range: TBR
mean
= 0.29 - 0.62; TBR
max
= 0.49 - 1.10) and highest in the HFHC group: TBR
mean
= 0.66 and TBR
max
= 1.28 (range: TBR
mean
= 0.26 - 1.38; TBR
max
= 0.39 - 3.19). Plasma lipids: lean control: TC = 1.34 mmol/L; TG = 0.38 mmol/L; HFHC group (mean values): TC = 18.7mmol/L (range: 11.6 - 25.8mmol/L); TG = 0.6 mmol/L (range: 0.37 - 0.83mmol/L).

Conclusion:
Non-invasive
64
Cu-DOTATATE PET/MR is feasible for assessment of SSTR
2
expression in a Göttingen minipig model of diet-induced atherosclerosis. Additional studies are needed including assessment of histology findings and gene expression to confirm the presence of activated macrophages in order to validate the use of
64
Cu-DOTATATE PET/MR in this model.
Abstract 461: Hepatic Nuclear Factor 4 Alpha as a Regulator of Alanine: Glyoxylate Aminotransferase 2 Expression and Systemic Levels of Endogenous Methylarginines
Authors: Rodionov Roman N , Burdin Dmitri V , Kolobov Alexey A , Demyanov Anton V , Soshnev Alexey A , Brocker Chad , Samusik Nikolay , Brilloff Silke , Jarzebska Natalia , Martens-Lobenhoffer Jens , Reetz Theresa , Maas Renke , Bode-Böger Stefanie M , Gonzalez Frank , Weiss Norbert
Published: June 2021

Introduction:
Endogenous methylarginines have been proposed as markers and potentially mediators of cardiovascular diseases. Alanine:glyoxylate aminotransferase 2 (AGXT2) is the only enzyme capable of regulation of plasma levels of all three endogenous methylarginines. It has also been demonstrated that AGXT2 and its alternative substrate beta-aminoisobutyric acid (BAIB) can play an important modulatory role in lipid metabolism. Using bioinformatic analysis we identified a highly conserved putative binding site for the diabetes-associated transcription factor hepatic nuclear factor 4 alpha (HNF4A) in the mammalian AGXT2 promoter region. The aim of this study was to test the hypothesis that HNF4a is the major regulator of AGXT2 expression and activity.

Methods and results:
We introduced several point mutations in the putative HNF4A binding site and investigated their influence on activity of the murine
Agxt2
promoter using luciferase reporter assay. The mutated constructs decreased the activity of the reporter gene by 75% as compared to the native promoter sequence. We showed direct binding of HNF4a to
Agxt2
promoter using chromatin immunoprecipitation. We were able to demonstrate that siRNA-mediated knockdown of HNF4a leads to 50% reduction of
Agxt2
expression in the murine hepatic cell line Hepa 1-6. In the
in-vivo
part of the project we showed that liver-specific
Hnf4a
knockout mice have a 90% reduction in liver
Agxt2
mRNA levels, a 85% decrease in liver AGXT2 activity and significantly increased plasma levels of endogenous methylarginines and BAIB.

Conclusions:
In the current study we showed direct binding of HNF4a to the mammalian AGXT2 promoter region. We also demonstrated using
in-vitro
and
in-vivo
approaches that HNF4A is the major regulator of
Agxt2
expression and has direct influence on systemic levels of endogenous methylarginines and BAIB. These findings suggest a novel link between NO-mediated impairment of vascular and renal function and lipid metabolism.
Abstract 467: Lipid Phosphate Phosphatase 3 in Smooth Muscle Cells Suppresses the Development of Atherosclerosis
Authors: Yang Liping , Mueller Paul , Sunkara Manjula , Morris Andrew , Smyth Susan
Published: June 2021

Lipid phosphate phosphatase3 (LPP3) encoded by the PPAP2B gene, is an integral membrane enzyme that degrades bioactive lysophospholipids including lysophosphatidic acid (LPA), sphingosine-1-phosphate (S1P) and related bioactive lipids. These bioactive lipids regulate a wide variety of cellular functions such as proliferation, apoptosis and differentiation. We suggest that LPP3 hydrolysis of LPA is a critical step in regulating pathophysiological important responses in vascular cells. To test this hypothesis, we generated mice lacking LPP3 by breeding
Ppap2B

fl

/

fl

mice (fl/fl) animals expressing Cre recombinase under control of the SM22 promoter on the LDLr-/- background. At weaning, littermate SM22-Δ and fl/fl mice were fed Western Diet (WD) for 12 weeks. LPP3 expression in aortic SMCs was reduced (0.139 ± 0.0246 vs 1.0 ± 0; P < 0.001) and LPA content was significantly higher (9.32 ± 4.66 vs 5.66 ± 2.65; P < 0.05) in the aortic arch in SM22-Δ mice compared to control fl/fl mice. SM22-Δ mice demonstrated increased atherosclerotic lesions (18.98 ± 7.11% vs 6.06 ± 4.01%; P < 0.001), measured by en face analysis, of the aorta and significantly higher IL-6 expression (18.83 ± 2.87 vs 3.59 ± 8.92; P < 0.05) in the aortic arch. Sections of aorta at the level of the aortic root indicated that the loss of LPP3 upregulate CD68 and α-SMA staining. These results reveal that LPP3 normally functions to suppress atherosclerosis. LPP3 deficiency increases vascular LPA content, may promote the infiltration of inflammatory cells as macrophages into lesions and smooth muscle cell proliferation to accelerate the development of atherosclerosis.
Abstract 473: Identification of Apolipoproteins Using Feature Selection Technique
Authors: Tang Hua , Lin Hao
Published: June 2021

Objective:
Apolipoproteins are of great physiological importance and are associated with different diseases such as dyslipidemia, thrombogenesis and angiocardiopathy. Apolipoproteins have therefore emerged as key risk markers and important research targets yet the types of apolipoproteins has not been fully elucidated. Accurate identification of the apoliproproteins is very crucial to the comprehension of cardiovascular diseases and drug design. The aim of this study is to develop a powerful model to precisely identify apolipoproteins.

Approach and Results:
We manually collected a non-redundant dataset of 53 apoliproproteins and 136 non-apoliproproteins with the sequence identify of less than 40% from UniProt. After formulating the protein sequence samples with
g
-gap dipeptide composition (here
g
=1~10), the analysis of various (ANOVA) was adopted to find out the best feature subset which can achieve the best accuracy. Support Vector Machine (SVM) was then used to perform classification. The predictive model was evaluated using a five-fold cross-validation which yielded a sensitivity of 96.2%, a specificity of 99.3%, and an accuracy of 98.4%. The study indicated that the proposed method could be a feasible means of conducting preliminary analyses of apoliproproteins.

Conclusion:
We demonstrated that apoliproproteins can be predicted from their primary sequences. Also we discovered the special dipeptide distribution in apoliproproteins. These findings open new perspectives to improve apoliproproteins prediction by considering the specific dipeptides. We expect that these findings will help to improve drug development in anti-angiocardiopathy disease.

Key words:

Apoliproproteins

Angiocardiopathy

Support Vector Machine
Abstract 51: Allograft Inflammatory Factor-1 Deficiency Prevents Obesity and Insulin Resistance by Promoting Brown and Beige Adipocyte Thermogenic Programs
Authors: Chinnasamy Prameladevi , Srinivasan Aparna , Casimiro Isabel , Riascos-Bernal Dario F , Sibinga Nicholas E
Published: June 2021

Objectives:
Human population studies link sequence variants near the allograft inflammatory factor (Aif)-1 locus with obesity, adipose inflammation, and diabetes, conditions strongly associated with cardiovascular disease. We assessed possible causal roles of Aif-1 in these conditions using mouse models.

Hypothesis:
Aif-1 promotes obesity, adipose inflammation, and glucose intolerance.

Methods:
We generated Aif-1-deficient (

aif-1
-/-

) mice and fed them a normal or high fat diet (ND or HFD, respectively) for 16 weeks. Body composition was analyzed by microCT scan, tissues by macroscopic and microscopic methods to assess morphology, and gene expression by qRT-PCR. Metabolic activity was measured in metabolic chambers. Wt littermates served as controls.

Results:
Aif-1 was expressed in visceral, subcutaneous, and brown adipose tissues (VAT, SAT, and BAT, respectively).

Aif-1
-/-

mice showed less HFD-induced gain in body weight (
wt
vs.

aif-1
-/-

; 20 vs. 7.5g, p≤0.01) and fat mass (
wt
vs.

aif-1
-/-

; 21 vs.10g, p≤0.001), while lean mass did not change. We also found reduced tissue mass in VAT (
wt
vs.

aif-1
-/-
;

1.7 vs. 0.7g, p≤0.01), SAT (
wt
vs.

aif-1
-/-
;

2.5 vs. 0.4, p ≤0.0001) and BAT (
wt
vs.

aif-1
-/-

; 0.19 vs. 0.07, p≤0.01), with smaller adipocytes in both VAT and SAT. In addition, reduced lipid accumulation in BAT and liver was seen in

aif-1
-/-

mice on HFD compared to
wt
controls. Interestingly,

aif-1
-/-

mice showed increased metabolic activity (energy expenditure,
wt
vs.

aif-1
-/-

; 14 vs.16 Kcal/kg/h, p≤0.05), while physical activity was unchanged. At the molecular level,

aif-1
-/-

mice showed increased expression of uncoupling protein-1 in BAT, SAT, and VAT, as well as its co-activators PGC1α and PRDM16 in BAT and SAT. These findings suggest increased brown and beige adipogenesis in BAT and SAT, respectively. Compared to controls,

aif-1
-/-

mice were also more glucose tolerant and insulin-sensitive on either ND or HFD, and this correlated with macrophage polarization toward an M2 vs. M1 phenotype in VAT.

Conclusion:
Our data suggest that Aif-1 deficiency prevents HFD-induced obesity by enhancing metabolic activity via increased brown or beige adipocyte programs, which reduces inflammation and promotes glucose tolerance and insulin sensitivity.
Abstract 519: Therapeutic Angiogenesis by Peripheral Blood Mononuclear Cells and its Rationale for Designing a New Trial
Authors: Tateno Kaoru , Fujimoto Yoshihide , Hasegawa Hiroshi , Nagai Toshio , Kobayashi Yoshio
Published: June 2021

Background:
Clinical trials of therapeutic angiogenesis that treat chronic critical limb ischemia (CLI) are revealing mixed results. This may partly be due to unapt patient selection or suboptimal study protocols. Here, from our registry, we report demographic and procedural characteristics of patients who underwent major limb amputation despite therapeutic angiogenesis.

Methods:
Registry of CLI patients who were treated by peripheral blood mononuclear cell (PBMNC) implantation was retrospectively analyzed. Cases that underwent major limb amputation within 6 months of the therapy were further investigated to clarify any features that might have affected their limb outcomes.

Results and Discussions:
Among total of 112 patients in the registry, eight cases lost to follow up and were excluded from this study. Sixteen patients (16/104, 15.4%) underwent major limb amputation within 6 months after therapeutic angiogenesis. Six amputees had active infection in the treated limb. Unlike excellent outcome by bone marrow derived cells, any patients with systemic sclerosis treated by PBMNC had poor limb salvage. However, half (8/16) of the amputees had experienced blood flow recovery, at least for transient period of two weeks. Among these patients, five limbs showed significant recovery of crural blood flow, while in the ipsilateral foot, distal blood flow deteriorated, indicating the flow steal phenomenon induced by cell therapy, which induces strong vasodilatation. Excluding patients with active infection or systemic sclerosis halved the limb amputation rate. Careful selection of sites to implant cells according to anatomical distribution of arterial blood flow could even have improved the limb salvage rate.

Conclusion:
When designing a clinical trial of therapeutic angiogenesis, (1) excluding patients with active infections, (2) proper treatment site considering anatomical distribution of arterial blood flow to avoid steal phenomenon, and (3) selection of cell source according to etiology of CLI, may clarify the efficacy of therapeutic angiogenesis.
Abstract 520: Should Bilateral Internal Thoracic Artery Grafting be Used in Patients with Peripheral Vascular Disease?
Authors: Teich Nadav , Medalion Benjamin , Pevni Dmitry , Mohr Rephael , Kramer Amir , Paz Yosef , Nesher Nachum , Ben-gal Yanai
Published: June 2021

Background:
The potential survival benefit of Bilateral Internal Thoracic Artery (BITA) compared to Single Internal Thoracic Artery (SITA) grafting in peripheral or cerebral vascular disease (PVD) patients is questionable, due to their short life expectancy and increased risk of sternal wound infection.

Methods:
Six hundreds and four Patients with PVD who underwent BITA grafting between 1996 and 2010 were compared with 478 PVD patients who underwent SITA grafting.

Results:
Patients undergoing SITA were older, more often female, more likely to have chronic obstructive lung disease, EF<30% ,Diabetes, renal insufficiency, congestive heart failure and emergency operation. Euroscore of SITA patients was significantly higher(10.1 ±3.1vs. 8.1± 3.3 %,p<0.001) Operative mortality (4.4% vs. 5.0% in BITA and SITA)and sternal wound infections (4.4% vs. 3.6%) were not significantly different between groups. Median follow-up was 9.50 (95%CI 8.83-10.16) years. Ten-year survival (Kaplan-Meier ) of the SITA and BITA groups were not significantly different (45.1±4.7% vs. 50.1±3.4%,
P
=0.736, Log Rank test) and assignment to the BITA group was not associated with better propensity-adjusted survival (HR 1.050, 95% CI: 0.875-1.261,
P
=0.600) ( Stratified COX model. )

Conclusions:
This study shows. that, early and long-term outcomes of BITA grafting in patients with PVD are not better than those of SITA grafting. Early mortality from non-cardiac causes ,reduces the influence of the type of conduit used(BITA or SITA) on survival. Selective use of BITA in lower-risk PVD patients might un-mask the benefits of BITA grafting
Abstract 521: Janus Kinase 3 is a Novel Regulator for Smooth Muscle Proliferation and Vascularremodeling
Authors: Wang Yung-Chun
Published: June 2021

Vascular remodeling due to smooth muscle cell (SMC) proliferation is a common process occurring in a number of vascular diseases such as atherosclerosis, aortic aneurysm, posttransplant vasculopathy, and restenosis after angioplasty, etc. The molecular mechanism underlying SMC proliferation, however, is not completed understood. Our present study has identified Janus kinase 3 (JAK3), a member of the Janus kinase family, as a novel regulator for SMC proliferation. Platelet-derived growth factor (PDGF)-BB, a SMC mitogen, induces JAK3 expression and phosphorylation while stimulating SMC proliferation. Janex-1, a specific inhibitor of JAK3, or knockdown of JAK3 by shRNA inhibits SMC proliferation. Conversely, ectopic expression of JAK3 promotes SMC proliferation. Interestingly, JAK3 does not affect SMC contractile protein expression, suggesting that JAK3 mediate the proliferation, but not the phenotypic modulation of SMC. Mechanistically, JAK3 promotes SMC proliferation by regulating phosphorylation of Signal transducer and activator of transcription 3 and c-Jun N-terminal kinase. In vivo, by using a rat carotid balloon-injury model, we found that knockdown of JAK3 significantly attenuates injury-induced neointima formation. Importantly, JAK3 knockdown blocked the expression of proliferating cell nuclear antigen, suggesting that JAK3 is essential for SMC proliferation in vivo. Moreover, JAK3 knockdown prompts apoptosis in neointima SMC, indicating that JAK3 also stimulates SMC survival during neointima formation. Collectively, our data demonstrate that JAK3 mediates vascular remodeling by promoting both the SMC proliferation and survival.
Abstract 523: Factor XIIa: New Insights on Chemical Tractability and Target Indications From a Potent and Selective Tool Inhibitor
Authors: Barbieri Christopher M , Wang Xinkang , Zhou Xueping , Ogawa Aimie M , O'Neil Kim , Wu Weizhen , Castriota Gino , Seiffert Dietmar A , Gutstein David , Chen Zhu
Published: June 2021

FXII is an emerging target for thrombosis, yet several questions remain to be addressed. Firstly, from drug discovery perspective, level of enzyme occupancy needed for efficacy (which largely dictates potency and selectivity requirement for small molecule inhibitors) is unclear, as most reported active site inhibitors have some level of off-target activities. Secondly, from disease treatment perspective, it is unclear whether FXIIa inhibition will be a safe strategy for stroke treatment or prevention, as it was recently reported that FXIIa inhibition destabilized the subocclusive thrombi in a plaque rupture model. In this presentation, we set out to address these questions using a previously described molecule, Inf4mut15. We generated the human albumin (HA)-Inf4mut15 fusion protein (Mut-inf) for our studies. In vitro Mut-inf displayed comparable potency as the widely used wild-type HA-Infestin4 (WT-inf) (human FXIIa Ki = 73 and 120 pM, respectively). Both infs acted as competitive reversible active site inhibitors of FXIIa, with no binding to FXII zymogen, hence same mode of action as certain small molecule inhibitors. Mut-inf, however, was much more selective against plasmin compared to WT-inf (20,000- and 75-fold Ki separation, respectively), consistent with results from the functional tPA-induced TEG assay, where Ly60 was reduced dose-dependently by WT- but not Mut-inf. Mut-inf aPTT doubling concentration was 15 uM and FXIIa Ki in 30% plasma was 3.5 nM. Calculated enzyme occupancy for Mut-inf for doubling human aPTT is thus 99.9%. In the rabbit model of cerebral microembolic signals (MES) induced by FeCl
3
injury of the carotid artery, treatment with vehicle (n=7), WT-, and Mut-inf (1mg/kg and n=5 each) produced arterial thrombus of 6.0±0.4, 1.9±0.6, and 0.2±0.1 mg, respectively; incidence of MES detected in the middle cerebral artery was 4.1±1.3, 1.8±0.6, and 0.0±0.0, respectively. In summary, our studies demonstrated that very high enzyme occupancy will be required for achieving a putative aPTT doubling target in human for FXIIa active site inhibitors, highlighting the challenge with the small molecule modality. Our MES studies suggest that targeting FXII may offer a safe strategy for stroke prevention and/or other thromboembolic disorders.
Abstract 527: Vorapaxar Does Not Increase Bleeding Time
Authors: Kraft Walter , Gilmartin Jocelyn , Chappell Derek L , Nagalla Srikanth , Naik Uhlas P , Wrishko Rebecca E , Zhang Sandra , Anderson Matt S
Published: June 2021

Vorapaxar is a novel PAR-1 inhibitor approved in the US and EU to reduce risk of thrombotic CV events in patients with a history of MI, and in the US also in patients with PAD. It does not affect coagulation tests (TT, PT, aPTT, ACT, ECT). Aspirin and P2Y
12
inhibitors prolong human bleeding time (BT); does vorapaxar?

Methods:
In this randomized, active controlled, parallel group, 2-period, single-blind, open-label trial, healthy men (n=31) and women (n=5), in Period 1, received either 81 mg aspirin (ASA) QD for 7 days (N=18), or a 7 day regimen of vorapaxar (N=18) achieving steady state plasma concentrations equivalent to chronic 2.5 mg QD doses. In Period 2, each group added 7 days of the therapy alternate to that of Period 1 without washout. BT and platelet aggregation (PA) using arachidonic acid, ADP, and TRAP agonists were collected predose in Periods 1 (baseline) and 2, and 24 h after Period 2 last dose. A linear mixed effects model with fixed effect for treatment analyzed data.

Results:
BT geometric mean ratio (90% CI) for (vorapaxar/baseline) was 1.01 (0.88, 1.15), (ASA/baseline) was 1.32 (1.15, 1.51), (vorapaxar+ASA/vorapaxar) was 1.47 (1.26, 1.70), (vorapaxar+ASA/ASA) was 1.12 (0.96, 1.30). Each antiplatelet inhibited PA only as expected. See figure.

Conclusions:
Unlike ASA, vorapaxar did not prolong BT compared to baseline. When given with ASA, there is a slight numerical increase in BT beyond that of ASA. Implications for clinical spontaneous or surgical bleeding await further analyses of clinical trial data.
Abstract 543: Enhancement of Rat Lymphatic Lipid Transport by Glucose Ingestion
Authors: Hayashi Hiroshi , Kawamura Mitsunobu , Seishima Mitsuru
Published: June 2021

The intestine is a major source of lipid in the body. The mechanisms of absorption, intracellular metabolism, and lymphatic transport of intestinally derived lipid have been investigated vigorously. The lipid in the intestinal lumen is supplied as bile and food, the latter of which usually contains many kinds of nutrients. Although the intestinal lipid absorption physiologically occurs with the absorption of other nutrients, little is known about the effect of simultaneously absorbed other nutrients on lipid absorption in the intestine. The aim of this study is to clarify the effect of glucose ingestion on lipid transport in rat mesenteric lymph. The glucose is derived from carbohydrate which is a major constituent of food. A mesenteric lymph fistula was made in male Sprague-Dawley rats and the duodenum was cannulated through the fundus of stomach. Postoperatively, the rats were allowed to recover in restraint cages and intraduodenally infused with saline. On the day after surgery, the saline infusion was replaced by a lipid infusion containing 35.4 mg/h of triolein, added with cholesterol, egg lecithin, and taurocholate, emulsified in phosphate-buffered saline with (the experimental group) or without (the control group) 10% glucose. While the lipid emulsion was infused for 8 hours, lymph was collected before and hourly during the lipid infusion. The concentrations of glucose, triglyceride (TG), cholesterol, and phosphatidylcholine (PC) in lymph were measured enzymatically. The glucose concentration in lymph increased in the experimental group. The lymphatic glucose concentrations in 8 h were 338 ± 79 (mean ± SE) mg/dl in the experimental group (n = 5) and 152 ± 14 mg/dl in the control group (n = 4), respectively. The amount of TG transported in lymph for 8 hours were 175 ± 3 mg in the experimental group and 147 ± 7 mg in the control group, respectively, with a statistically significant difference (p < 0.01). PC was also transported in lymph significantly more in the experimental group than in the control group. In conclusion, simultaneous glucose feeding enhanced lymphatic lipid transport in the rat intestine.
Abstract 549: ABCA1 Expression Promotes ApoAI Acidification on the Plasma Membrane via Recruitment of Vacuolar ATPase
Authors: Wang Shuhui , Brubaker Gregory , Smith Jonathan D
Published: June 2021

Objective:
We have shown that ABCA1 mediates unfolding of apoAI N-terminal helical hairpin on the cell surface. Others have shown that acidic pH exposes apoAI’s hydrophobic surfaces. We postulated that ABCA1 may mediate the local acidification of the plasma membrane to promote apoAI unfolding and lipidation.

Methods and Results:
We found that apoAI unfolding in guanidine was facilitated at acidic pH (EC
50
for unfolding at 0.94 and 0.75 M guanidine at pH 7.5 and 5, respectively), and confirmed that acidic conditions promoted apoAI solubilization of and binding to egg PC:PS (8:2 mole ratio) liposomes. We generated a FITC/Alexa647-labeled fluorescent ratiometric apoAI pH indicator whose FITC/Alexa647 emission ratio decreased as pH decreased. Using flow cytometry in BHK and RAW264.7 cell lines, we found that incubation of the apoAI pH indicator with ABCA1 expressing cells at 37
o
C led to substantial acidification of apoAI, which may be due to the uptake of apoAI into endosomes/lysosomes. However, incubation of the apoAI pH indicator with ABCA1 expressing cells at 21
o
C or with an inhibitor of endocytosis, where apoAI is on the cell surface, still led to mild apoAI acidification. To elucidate the mechanism of ABCA1 effects on local acidification of the plasma membrane, we examined if ABCA1 regulates vacuolar ATPase (V-ATPase) proton pump, which is composed of a peripheral V
1
domain and an embedded V
0
domain. We observed that bafilomycin, the V-ATPase inhibitor, eliminated the pH shift of apoAI on plasma membrane, and dose dependently inhibited ABCA1 mediated cholesterol efflux to apoAI. Although ABCA1 did not alter total cellular levels of V-ATPase, we showed that ABCA1 increased the cell surface levels of the V
0
A1 and V
1
E1 subunits of V-ATPase by cell surface biotinylation and immunofluorescence. Moreover, inhibition of apoAI acidification by bafilomycin decreased apoAI unfolding on the cell surface.

Conclusions:
Our results support that ABCA1 induces the levels of V-ATPase on the cell surface leading to local acidification of apoAI, which promotes apoAI unfolding and lipidation.
Abstract 55: Traf3 Negatively Regulates Platelet Activation and Thrombosis
Authors: Zhang Rui , Zhang Guoying , Xiang Binggang , Xie Ping , Li Zhenyu
Published: June 2021

Platelets express CD40 ligand (CD40L) and are a major source of soluble CD40L. CD40L has been shown to potentiate platelet activation and thrombus formation, involving both CD40-dependent and -independent mechanism. CD40L-mediated platelet activation requires the family of TNF receptor-associated factor 2 (TRAF2). Here we show that platelet also express TRAF3, which plays a negative role in regulating platelet activation. Thrombin- or collagen-induced platelet aggregation and secretion are increased in TRAF3 knockout mice. The expression of integrin αIIbβ3, GPVI, and PAR4 was similar between TRAF3 knockout platelets and wild type controls, suggesting that increased platelet activation in the TRAF3 knockout mice is not due to increased expression of platelet receptors. TRAF2 expression was not affect by deletion of TRAF3 eitehr. Using the FeCl
3
-induced arterial thrombosis model, we found that time to formation of thrombi in was significantly shortened in TRAF3 knockout mice. Thus, TRAF3 plays a negative role in platelet activation and in thrombus formation in vivo.
Abstract 551: Loss of ABCG1 Specifically in Pulmonary Type 2 Cells Results in Impaired Surfactant Lipid Metabolism
Authors: de Aguiar Vallim Thomas Q , Merriott David J , Cheng Joan , Cheng Angela , Baldan Angel , Ford David A , Goulbourne Christopher N , Tarling Elizabeth J
Published: June 2021

Cardiovascular disease (CVD) is the leading cause of death in the United States, and patients with pulmonary disease are at least 2-fold more likely to develop cardiovascular complications. Whole-body cholesterol balance is a complex and tightly regulated process. The sterol-transporter ATP Binding Cassette transporter G1 (ABCG1) is critical for maintaining normal cellular cholesterol and lipid homeostasis. After the liver and intestine, the lung is the 3rd most active lipid-secreting organ. Pulmonary alveolar proteinosis (PAP) is a rare lung disease characterized by the accumulation of surfactant lipids and proteins in the pulmonary alveoli resulting in respiratory distress. For almost all reported cases the underlying cause is unknown. Synthesis and secretion of surfactant in the lung is restricted to epithelial type 2 pneumocytes (T2 cells). However, subsequent clearance of surfactant from the hypophase is dependent upon both T2 cells and macrophages. Both T2 cells and macrophages express high levels of ABCG1. Abcg1-/- mice develop a severe pulmonary lipidosis, characterized by elevated levels of multiple lipid species and the presence of lipid-loaded macrophages and T2 cells containing abnormal lamellar bodies. Recent advances have also implicated a role for the innate immune system in the pathogenesis of PAP. We recently demonstrated that the lungs of Abcg1-/- mice exhibit a profound immune response associated with a local increase in innate immune natural antibody-secreting B-1 B cells and both local and circulating natural antibodies. Here we show that mice lacking ABCG1 specifically in T2 cells develop abnormal lamellar bodies with altered phospholipid and cholesterol homeostasis, and altered gene expression. Collectively, our results suggest that ABCG1 is critical for normal surfactant metabolism and that the pulmonary expansion of B-1 B cells and the subsequent increase in natural antibodies in Abcg1-/- mice may provide a novel model in which to study the role of the innate immune system in PAP, and the subsequent risk of developing cardiovascular disease.
Abstract 556: The Soluble Guanylyl Cyclase Activator Bay 60-2770 Inhibits Arterial Smooth Muscle Cell Migration in Protein Kinase G-dependent Manner
Authors: Holt Andrew , Martin Danielle , Shaver Patti , Adderley Shaquria , Stone Joshua , Joshi Chintamani , Francisco Jake , Lust Bob , Shewchuk Brian , Tulis David
Published: June 2021

Atherosclerotic lower extremity peripheral artery disease (PAD) is among the most prevalent, morbid and mortal of all cardiovascular disorders. Pathologic arterial smooth muscle (ASM) cell migration is a major component of atherogenic PAD and efforts aimed at attenuating its progression are clinically essential. Cyclic nucleotide signaling has long been studied for its growth-mitigating properties in the setting of PAD and other vascular growth disorders. In this study we hypothesized that the novel, heme-independent soluble guanylyl cyclase activator BAY 60-2770 (BAY) inhibits ASM cell migration through phosphorylation of the protein kinase G (PKG) target and actin-binding protein vasodilator-stimulated phosphoprotein (VASP). In a rat model of injury-induced arterial growth, BAY significantly reduced neointima formation and luminal narrowing compared to vehicle (Veh)-treated control arteries after 2 weeks. Using rat and human ASM cells BAY significantly attenuated cell migration, reduced G:F actin, and increased cyclic GMP content, PKG activity and phosphorylated VASP at Ser239 (pVASP.S239) compared to Veh controls. Using site-directed mutagenesis, both full-length VASP-overexpressing (wild type, WT) and VASP.S239 phosphorylation-resistant mutants showed significantly reduced cell migration compared to naïve controls, however, there was no effect on cell migration between either VASP transfected group in the presence of BAY. Interestingly, both VASP mutants showed significantly increased PKG activity compared to naïve cells, and in turn pharmacologic PKG blockade in the presence of BAY fully reversed the inhibitory effect of BAY alone on cell migration. These data suggest BAY has capacity to inhibit ASM cell migration through cyclic GMP/PKG/VASP signaling yet through mechanisms independent of pVASP.S239. Findings from this study implicate BAY via cyclic GMP/PKG/VASP as a potential pharmacotherapeutic agent against aberrant ASM growth disorders such as PAD.
Abstract 567: Transcription Factor Runx2 is Induced in Vascular Aging and May Promote Age-related Arterial Stiffness
Authors: Schellinger Isabel N , Spin Joshua M , Hasenfuss Gerd , Tsao Philip S , Raaz Uwe
Published: June 2021

Background:
Arterial stiffening is a hallmark of vascular aging and may constitute a critical mechanism linking age to increased cardiovascular risk. However, up to now there is no therapy available to efficiently and specifically target age-related arterial stiffening. We recently identified the osteogenic transcription factor Runx2 as an inducer of diabetic arterial stiffness.

Objective:
The present study investigated the role of Runx2 in the setting of age-related arterial stiffness.

Methods and Results:
Aortic stiffness – quantified by
ex vivo
mechanical testing (pressure myography) – was markedly increased in 1-year old male C57Bl/6 mice compared to young (10 week-old) controls. At the same time, Runx2 was aberrantly upregulated in the medial layer of aged aortae, coming along with significant medial fibrosis. Additionally, we detected increased aortic expression of interleukin 6 (
Il6
) – a key cytokine involved in vascular “inflammaging”. Mechanistically, we found IL-6-induced
RUNX2
expression in aortic smooth muscle cells (SMCs) via a NFkB-dependent pathway.

Conclusion:
In conclusion this study suggests Runx2 as a potential mediator of age-related arterial fibrosis and stiffness warranting further interventional/therapeutic studies.
Abstract 574: A Role for TWIST1 as GWAS Risk Gene for Coronary Artery Disease
Authors: Nurnberg Sylvia T , Testa Stephanie , Rosal Joebert , Elwyn Susannah , Zhao Wei , Rader Daniel J
Published: June 2021

Genome-wide association studies (GWAS) have identified rs2107595 as association SNP for both stroke and cardiovascular disease. This polymorphism is located proximal to the HDAC9 gene, with TWIST1 106kb downstream as the next closest gene in the recombination interval. Whilst studies in hyperlipidemic Hdac9 knockout mice have demonstrated a pro-atherogenic effect of Hdac9, recent evidence suggests an additionally role of TWIST1 in atherosclerosis. For one, rs2107595 genotypes associate with differences in TWIST1 but not HDAC9 expression levels in human aorta (GTEx consortium, n=197, p=0.00016), proposing a role of TWIST1 downstream the association locus specifically in vascular smooth muscle cells. RNA-seq data from
in vitro
cultured human coronary artery smooth muscle (HCASMCs) and endothelial cells (HCAECs) shows differential expression of TWIST1 in smooth muscle cells (n=19/20, q=3E-60). Furthermore, TWIST1 expression is selectively upregulated in HCASMCs upon serum starvation, and TWIST1 protein is detected in human lesions both near the tunica media and at the fibrous cap where it co-localizes with smooth muscle actin (ACTA2).

The GWAS association SNP is predicted to change transcription factor binding affinity from the more general E2F to RBPJ - a well-known modulator of Notch signaling in smooth muscle cells (Transfac professional, 2014.4 data release). The variant is located within an enhancer region in mesenchymal stem cells (Broad GSE17312), fibroblasts (UCSF-UBC-USD GSE16368) as well as in HCASMCs.

Current studies explore the tissue-specific activity of this regulatory element through
in vivo
lacZ reporter assays to investigate a potential role in mouse coronary arteries. Genome editing studies in immortalized HCASMCs are being employed to investigate the effect of SNP genotype on expression levels of both TWIST1 and HDAC9. Future experiments using an inducible, smooth muscle specific knockout of Twist1 will interrogate the potential role of Twist1 in atherosclerosis.
Abstract 581: Divergent Roles for Mertk and Axl in Resident Vs Recruited Phagocytes After Reperfusion of Ischemic Hearts
Authors: DeBerge Matthew , Yeap Xin Yi , Procissi Daniel , Tabas Ira , Thorp Edward
Published: June 2021

Objective:
Following myocardial infarction (MI), recruited and resident phagocytes are mobilized at the heart to promote clearance of dying myocardial cells by efferocytosis. This occurs in part through the actions of the macrophage receptor tyrosine kinases, Tyro 3, Axl, and Mertk (TAM), although potential differential contributions of TAMs on recruited vs resident phagocytes, as well as the contribution to clinically-relevant reperfusion, are unknown. We set out to test the significance of recruited vs resident Mertk
+
phagocytes during ischemia/reperfusion (I/R) and in comparison to Axl
+
phagocytes.

Methods and Results:
In contrast to MI, I/R resulted in elevated inflammation and early loss of Mertk on phagocytes in the infarcted tissue with a concomitant increase in serum levels of soluble Mertk in both mice and humans. In mice, Mertk-deficiency during I/R led to decreased cardiac efferocytosis, increased infarct size, and depressed cardiac contractility, newly revealing the importance of efferocytosis during clinically-relevant reperfusion. These endpoints were similarly affected after blocking blood-borne recruited monocytes with CCR2 antagonists, thereby specifically implicating resident Mertk
+
phagocytes to cardiac repair. We observed the highest expression of Mertk on Ly6C
lo
MHCII
lo
cardiac resident macrophages, which resulted in increased efferocytosis-efficiency by these cells compared to other cardiac phagocytes. Shedding of Mertk from Ly6C
lo
MHCII
lo
cardiac resident macrophages was observed after I/R and specific inhibition of Mertk cleavage using mice that express a cleavage-resistant Mertk reduced infarct size after I/R, identifying Mertk cleavage as a new therapeutic target after reperfusion injury. Interestingly, Axl was selectively expressed on Ly6C
lo
MHCII
hi
cardiac resident macrophages and surprisingly, Axl-deficiency during I/R led to reduced infarct size and improved cardiac contractility, suggesting differential roles between TAMs during cardiac repair.

Conclusions:
These data newly support the importance of efferocytosis pathways post-I/R and reveal both Axl and specifically Mertk on resident macrophages as key molecular mediators of inflammation resolution during atherothrombosis.