bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 314: H19 Induces Abdominal Aortic Aneurysm Development and Progression
Authors: Li Daniel Y , Busch Albert , Jin Hong , Chernogubova Ekaterina , Pelisek Jaroslav , Eriksson Per , Roy Joy , Hofmann Patrick , Boon Reinier , Spin Joshua , Tsao Philip , Maegdefessel Lars
Published: March 2019

Background:
Long noncoding RNAs (lncRNAs) have emerged as critical molecular regulators in various biological processes and diseases. Here we sought to identify and functionally characterize lncRNAs as potential mediators in abdominal aortic aneurysm (AAA) development.

Methods and results:
We profiled RNA transcript expression in two murine AAA models, Angiotensin II (ANGII) infusion in
ApoE-/-
mice (
n
=10) and porcine pancreatic elastase (PPE) instillation in C57BL/6 wildtype mice (
n
=12). The lncRNA H19 was identified as one of the most highly up-regulated transcripts in both mouse aneurysm models compared to sham-operated controls. This was confirmed by qRT-PCR and
in situ
hybridization. Experimental knock-down of H19, utilizing site-specific antisense oligonucleotides (LNA-GapmeRs)
in vivo
, significantly limited aneurysm growth in both models. Upregulated H19 correlated with smooth muscle cell (SMC) content and SMC apoptosis in progressing aneurysms. Importantly, a similar pattern could be observed in human AAA tissue samples, and in a novel preclinical
LDLR-/-
Yucatan mini-pig aneurysm model.
In vitro
knock-down of H19 markedly decreased apoptotic rates of cultured human aortic SMCs, while overexpression of H19 had the opposite effect. Notably, H19-dependent apoptosis mechanisms in SMCs appeared to be independent of miR-675, which is embedded in the first exon of the H19 gene. A customized transcription factor array identified hypoxia-inducible factor 1-alpha (HIF1α) as the main downstream effector. Increased SMC apoptosis was associated with cytoplasmic interaction between H19 and HIF1α and sequential p53 stabilization. Additionally, H19 induced transcription of HIF1α
via
recruiting specificity protein 1 (Sp1) transcription factor to the promoter region.

Conclusions:
The lncRNA H19 is a novel regulator of SMC survival in AAA development and progression. Inhibition of H19 expression might serve as a novel molecular therapeutic target for aortic aneurysm disease.
Abstract 316: Chronic Intermittent Hypoxia Attenuates BAPN Induced Thoracic Aortic Aneurysms in Mice
Authors: Yang Yunyun , Li Linyi , Qin Yan Wen
Published: March 2019

Objective:
Aortic aneurysm (AA) is a life-threatening cardiovascular disorder due to the predisposition for dissection and rupture. Chronic intermittent hypoxia (CIH) has protective effects on heart and brain against ischemia injury. However, whether CIH prevents against AA formation was not elucidated. The present study aimed to investigate the effect of CIH treatment on thoracic aortic aneurysms (TAA) in mice.

Approach and Results:
Seventy-five C57BL/6 mice 3 weeks of age were divided into three groups, i.e. Control group ( 21% O
2
, 24 h per day, 4 weeks, n = 15); β-aminopropionitrile (BAPN) group (oral administration BAPN 1g / kg / day, 21% O
2
, 24 h per day, 4 weeks, n = 30); CIH+BAPN group ( 5% -21% O
2
90s / cycle, 8h per day, 4 weeks; BAPN 1g / kg / day, n = 30). We found that CIH significantly reduced the incidence of TAA (18 of 30 versus 9 of 30; p<0.05), and decreased maximal aortic diameter in BAPN group by hematoxylin and eosin staining (1.71 ± 0.35 versus 1.23± 0.15 mm; p <0.05). CIH significantly reduced elastin breaks in BAPN group by Verhoeff-van Gieson staining. The apoptosis of vascular smooth muscle cells was detected by TUNEL staining increased in BAPN group and markedly reversed by CIH. Moreover, RNA-Seq analysis and bioinformatics analysis were performed to screen the differential expression genes and possible pathway.

CIH significantly down-regulated the expression of inflammation signaling pathways, up-regulated the expression of PI3K/AKT survival pathway and extracellular matrix homeostasis related pathway in BAPN induced TAA mice. Confirmed by real-time quantitative reverse-transcription-polymerase chain reaction (qRT-PCR) assays, CIH significantly inhibited the expression of IL-1β, TNF-α, MMP-9 and Cathepsin S, and increased the expression of hypoxia inducible factor-1α (HIF-1α), Lysyl oxidase (LOX), ITGB1 and COL1A2. Our findings also showed that the expression of MMP-9, MMP-2, cathepsin S, IL-1β, MCP-1 and Mac-2 increased in BAPN group and markedly decreased in CIH+BAPN group by immunohistochemistry staining. Besides, CIH also significantly increased the protein expression of HIF-1α, LOX and p-AKT by western blot analysis.

Conclusions:
The findings from our study suggest that CIH prevented the TAA development of BAPN-induced mice.
Abstract 319: Novel Method to Evaluate Nature of Vessel Wall in Abdominal Aortic Aneurysm by Fluorescent Probes Reacting Specific Protease
Authors: Seo Akihiko , Akagi Daisuke , Kamiya Mako , Kuriki Yugo , Hoshina Katsuyuki , Urano Yasuteru
Published: March 2019

Background and Objective:
Evaluation of the vessel wall condition during vascular surgery is an essential and important for technical success. However, no optimal method has been recommended for the objective assessment of the condition, and evaluation and decision of the suture/anastomotic sites are governed by the surgeon’s experience during surgery. Fluorescence-based visualization is a promising method available for objective assessment; therefore, we aimed to use this technique to distinguish non-aneurysmal wall from an aneurysmal one. Various proteases are known to play a role in aneurysm formation. We synthesized over 250 kinds of aminopeptidase-activatable fluorescent probes. To optimize aneurysmal wall evaluation based on specific fluorescent activity, these probes were investigated.

Methods and Results:
Vessel wall specimens were clinically obtained during open surgery for the abdominal aortic aneurysm at the University of Tokyo Hospital. Initially, to screen these probes comprehensively, the vessel walls were homogenized and supernatants were obtained. After incubating each probe in the supernatant, the increment of fluorescence was measured (excitation at 485 nm and detection at 535 nm). Among the 250 probes investigated, 6 showed a difference in the fluorescent activity between aneurysmal and non-aneurysmal walls. Next, these 6 probes were applied to freshly resected vessel walls on the from luminal side in the form of a gel, and fluorescent images were obtained using the Maestro® imaging system (PerkinElmer, Waltham, MA). Two probes showed a > 2-fold stronger activity in aneurysmal walls than that observed in non-aneurysmal walls.

Conclusion:
These probes may be useful for the objective evaluation of the nature of vascular walls. We plan to use small fluorescent detectors for native aortic wall intraoperatively and investigate target proteases that react with these probes.
Abstract 322: Exploring the Role of Triggering Receptor Expressed on Myeloid Cells-1 in Experimental Abdominal Aortic Aneurysm
Authors: Vandestienne Marie , Joffre Jérémie , Giraud Andréas , Potteaux Stéphane , Laurans Ludivine , Tedgui Alain , Derive Marc , Mallat Ziad , Aït-Oufella Hafid
Published: March 2019

Introduction:
Non-syndromic Abdominal Aortic Aneurysm (AAA) is one of the leading causes of cardiovascular death in elderly men. AAA is characterized by extracellular matrix degradation, smooth muscle cell apoptosis and infiltration of inflammatory cells in the aortic wall. Monocyte recruitment particularly plays an important role in the pathophysiology of AAA. TREM (Triggering Receptor Expressed on Myeloid cells)-1, a receptor expressed by myeloid cells, is a key regulator of innate immune cell activity, and we hypothesized that it might be involved in AAA development.

Objective:
We aim to decipher the role of TREM-1 in the immuno-inflammatory response associated with AAA development and rupture.

Method:
To study the role of TREM-1 in AAA development, we used an experimental model of Angiotensin-II-induced AAA in

ApoE
-/-
Trem-1
-/-
and ApoE
-/-
Trem-1
+/+

mice. At day 0, 3, 7 and 28, we analyzed monocyte trafficking (flow cytometry), inflammatory cell infiltration within the aortic wall (immunostaining, qPCR), aortic wall remodeling (histology) and finally AAA development.

Results:
Angiotensin II infusion induced a 2-fold increased of TREM-1 expression on classical monocytes at day 3. TREM-1 deficiency (

ApoE
-/-
Trem-1
-/-

) abolished Angiotensin II-induced monocytosis at day 3 and prevented macrophage infiltration within the aorta at day 7. TREM-1 genetic deletion also limited pro-inflammatory response (
Il-1beta
and
Tnf-alpha
mRNA expression) and metalloprotease activity in the aortic wall and protected against elastin degradation at day 7. At day 28 (n=10/group), under angiotensin II infusion,

ApoE
-/-
Trem-1
-/-

mice are characterized by reduced mean aorta diameter (702+/-36 μm versus 1081+/-51 μm, p<0.01), less severe AAA disease (p<0.001, Chi-squared for a trend) when compared to

ApoE
-/-
Trem-1
+/+

mice. In addition, TREM-1 deficiency protected against lethal AAA rupture (28-Day survival 100% versus 82% p=0.05).

Conclusion:
We showed that TREM-1 deficiency reduced AAA development and rupture in an Angiotensin II-induced mouse model.
Abstract 323: Hydrogen Sulfide Limits the Development of Intimal Hyperplasia in a Mouse Model of Femoral Wire Injury and in Human Veins
Authors: Longchamp Alban , Yamagushi Natsumi , Lambelet Martine , Dubuis Celine , Saucy Francois , Corpataux Jean-Marc , Allagnat Florent , Déglise Sebastien
Published: March 2019

Objectives:
Mainstays of contemporary therapies for this arterial occlusive disease include angioplasties, stents, endarterectomies and bypass surgery. However, these treatments suffer from high failure rates due to re-occlusive vascular wall adaptations, namely intimal hyperplasia (IH). IH develops in response to endothelium injury, leading to inflammation, vascular smooth muscle cells (VSMC) dedifferentiation, migration and proliferation at the site of injury. Hydrogen sulfide (H
2
S) is a ubiquitous signaling gazotransmitter, which exhibits antioxidant, anti-inflammatory, and vaso-relaxant properties. Thus, we hypothesized that H
2
S could reduce IH formation.

Methods:
WT male C57BL6/J mice submitted to femoral wire injury surgery to induce IH were treated with an H
2
S donor (NaHS) in the drinking water. IH was measured 28 days post-surgery by histology. In addition, segments of great saphenous vein obtained from patients undergoing bypass surgery were maintained in culture
ex-vivo
for 7days in presence of various H
2
S donors (NaHS, GYY4137, diacyltrisulfide). Finally, primary human umbilical vein endothelial cells (HUVEC) and primary human VSMC were treated
in-vitro
with the same H
2
S donors to study cellular proliferation and migration.

Results:
NaHS treatment significantly reduced IH development in the mouse model of femoral wire injury (
Figure 1
). Similarly, the various H
2
S donors prevented the development of IH in vein segments
ex-vivo. In vitro
, the same H
2
S donors stimulated human endothelial cells (HUVEC) migration and proliferation, while inhibiting migration and proliferation of primary VSMC (
Figure 2
).

Conclusions:
Exogenous H
2
S prevents IH formation in mice
in-vivo
and in human veins
ex-vivo
. Importantly, H
2
S reduces VSMCs but stimulates ECs proliferation and migration. These data suggest that exogenous H
2
S therapy could be used in human to minimize IH, thus limiting vascular reconstruction failure.
Abstract 324: Molecular Imaging of High Risk Atherosclerotic Plaque Using Fibrin-Binding PET Probe
Authors: Izquierdo-Garcia David , Diyabalanage Himashinie , Ramsay Ian , Rotile Nicholas J , Mauskapf Adam , Choi Ji-Kyung , Witzel Thomas , Humblet Valerie , Brownell Anna-Liisa , Catana Ciprian , Caravan Peter , Ay Ilknur
Published: March 2019

Objectives:
There is a need for better stratification of atherosclerotic patients to identify individuals at high risk for a cardiovascular event. Two key hallmarks of plaque at high risk for rupture are intraplaque hemorrhage and evidence of prior rupture. The protein fibrin is strongly associated with both hemorrhage and rupture but it is not present in earlier stages of plaque development, suggesting that fibrin imaging would be sensitive for high risk plaque versus stable/early disease. Here, we describe imaging properties of a fibrin-specific PET probe for atherosclerotic plaque rupture in rabbits.

Methods:
Rabbits were fed with high cholesterol diet (HCD) for 8 weeks followed by 2 weeks of normal diet. Two weeks after the initiation of HCD, endothelial denudation of abdominal aorta was performed using balloon catheter. At 10 weeks, rabbits were randomly divided into plaque rupture (n=8; triggered by Russell’s viper venom and histamine) and control group (n=4), injected with fibrin-binding probe [
68
Ga]CM-246, and scanned on a PET/MRI. Then, vessels of interest were removed for ex-vivo PET, autoradiography, and histology.

Results:
Rabbits with plaque rupture had >2-fold increase in [
68
Ga]CM-246 signal in the abdominal aorta compared with the control group (A). At 105-120 min post probe injection, there was a clear PET uptake on the aorta in plaque rupture rabbit (B) compared with control rabbit (C). MR images were used to differentiate abdominal aorta (green arrows) and the inferior vena cava (blue arrows). T2-dark blood images confirmed the atherosclerotic plaques in plaque rupture (D) and control rabbits (E). ToF images were used to visualize aortic lumen in plaque rupture (F) and control rabbits (G). Ex vivo PET imaging (H) and autoradiography confirmed in vivo results.

Conclusions:
We demonstrated that [
68
Ga]CM-246 detects atherosclerotic plaque rupture in a rabbit model and can be a useful tool for the diagnosis of high-risk atherosclerotic plaque in patients.
Abstract 330: Targeted Nanotherapy for the Treatment of Atherosclerosis
Authors: Mansukhani Neel A , So Miranda , Albaghdadi Mazen S , Peters Erica B , Wang Zheng , Stupp Samuel I , Kibbe Melina R
Published: March 2019

Objective:
Atherosclerosis is the leading cause of death and disability in the United States. We hypothesize that systemic administration of a novel nanofiber will target areas of atherosclerosis and regress atherosclerotic lesions.

Methods:
Self assembling peptide amphiphile (PA) nanofibers were synthesized. An 18 amino acid sequence which retains the cholesterol efflux actions of apolipoprotein-A1 (apoA1) along with a Liver X Receptor (LXR) agonist, known to enhance cholesterol efflux, were incorporated into the nanofiber to both target and treat atherosclerosis. To assess the ability of the nanofiber to target and treat atherosclerosis
in vivo
, LDL receptor knockout mouse (LDLR KO) mice were fed a high fat diet (HFD) for 14 weeks after which they received bi-weekly injections of the therapeutic or control for 8 weeks. Optimum dose, concentration, binding duration, and biodistribution was determined using fluorescent microscopy and pixel quantification. Treatment groups included: PBS control, PA nanofiber with a scrambled targeting sequence, LXR agonist alone, targeted PA nanofiber (ApoA PA), and targeted PA nanofiber incorporating LXR agonist (ApoA-LXR PA). n=10/treatment group.

Results:
ApoA PA and ApoA-LXR PA nanofibers effectively targeted atherosclerotic plaque in the aortic root. Optimum concentration of nanofiber was 2mg/mL, and optimum dose was 6mg/kg. There was no difference in optimum dosing or concentration between males and female mice. ApoA PA nanofiber localized to the aortic root for approximately 2-3 days, and was cleared from the aortic root by 7-10 days. Concentrations of ApoA PA in the aortic root was 5-fold higher than in the lung, liver, and kidney one day post injection. After only 8 weeks of treatment, male and female mice treated with ApoA-LXR PA had 11 and 9% plaque area reduction compared to PBS treated controls, respectively. Differences in treatment conditions vs. controls showed sex dependence, with only male mice demonstrating significantly higher plaque reduction from ApoA1-LXR PA treatment in comparison to scrambled PA treatment.

Conclusions:
Our results demonstrate that a novel targeted nanofiber binds specifically to atherosclerotic lesions and reduce plaque burden after a short treatment duration.
Abstract 333: Dynamic Limb-specific Metabolomics Reveals the Impact of Diabetes on Muscle and Vascular Responses to Ischemic Stress
Authors: Ferguson Jane F , Huang Shi , Gerszten Robert E , Beckman Joshua A
Published: March 2019

Background:
Type 2 Diabetes Mellitus (T2DM) increases cardiovascular risk, but the mechanisms are incompletely understood. Blood metabolomic signatures are altered in T2DM, but the impact of ischemic stress on the local skeletal muscle signature and T2DM-related vascular dysfunction is unknown.

Methods:
We recruited 38 subjects (18 healthy, 20 T2DM), placed an antecubital intravenous catheter, and performed ipsilateral brachial artery reactivity testing. Blood samples for plasma metabolite profiling were obtained at baseline and upon cuff release after 5 minutes of ischemia. Brachial artery diameter was measured at baseline and 1 minute after cuff release.

Results:
As expected, flow-mediated vasodilation was attenuated in subjects with T2DM (p<0.01). We confirmed known T2DM-associated baseline differences in plasma metabolites, including homocysteine, dimethylguanidino valeric acid and β-alanine (all p<0.05). Metabolites were significantly altered in response to experimental ischemia. Ischemia-induced metabolite changes that differed between groups included 5-hydroxyindoleacetic acid (Healthy: -27%; T2DM +14%), orotic acid (Healthy: +5%; T2DM -7%), trimethylamine-N-oxide (Healthy: -51%; T2DM +0.2%), and glyoxylic acid (Healthy: +19%; T2DM -6%) (all p<0.05). Serine, betaine, aminoisobutyric acid and anthranilic acid associated with vessel diameter at baseline, but only in T2DM (all p<0.05). Metabolite responses to ischemia were significantly associated with vasodilation extent, but primarily observed in T2DM. Within T2DM subjects, metabolite changes predicting change in vessel diameter included enrichment in amino acid, glycerophospholipid and propanoate metabolism (all p<0.05).

Conclusion:
Our study highlights potential impairments in muscle and vascular signaling at rest and during ischemic stress in T2DM. While metabolites change in both healthy and T2DM subjects in response to ischemia, a relationship between muscle metabolism and vascular function is modified in T2DM, suggesting that dysregulated muscle metabolism in T2DM may have direct effects on vascular function.
Abstract 334: Collateral Development in Swine after Ligation of Native Leg Arteries
Authors: Gao Yue , Aravind Shruthi , Fuglestad Matthew , Hansen Chris , Casale George , Carlson Mark , Pipinos Iraklis
Published: March 2019

Introduction:
The development of collateral vasculature is a key mechanism compensating for arterial occlusions in patients with Peripheral Artery Disease (PAD). We aimed to examine the development of collateral pathways after ligation of native vessels in a porcine model of PAD.

Methods:
Right hindlimb ischemia was induced in domestic swine (N=11, male, 26-57 kg) using two different versions of arterial ligation. Version 1 (N=6) consisted of ligation/division of the right external iliac, profunda femoral (RPFA) and superficial femoral arteries (RSFA). Version 2 (N=5) consisted of the ligation of Version 1 with additional ligation/division of the right internal iliac artery (RIIA). Development of collateral pathways was evaluated with standard angiography at baseline (prior to arterial ligation) and at termination (4-8 weeks later). Relative luminal diameter of the arteries supplying the ischemic right hindlimb were determined by 2D angiography, as percent of the size of the distal aortic diameter.

Results:
The pathway connecting the RIIA to the RPFA and RSFA/popliteal artery of the ischemic limb was the dominant collateral pathway in version 1. Mean luminal diameter (± standard error) of the RIIA at termination increased by 39.4± 5.5% (P<0.01) compared to baseline. There were two co-dominant collateral pathways in version 2. The first pathway connected the common internal iliac trunk and left internal iliac artery to the reconstituted RIIA which then supplied the RPFA and RSFA/popliteal arteries. The second pathway connected the left profunda artery to the reconstituted RPFA. Mean diameter (± standard error) of the common internal iliac trunk and left profunda artery increased at termination by 23.7± 7.6% and 24.8± 7.4%, respectively (p < 0.05).

Conclusion:
Two versions of hindlimb ischemia induction (right ilio-femoral artery ligation with and without right internal iliac artery ligation in swine produced differing collateral pathways along with changes to the diameter of the inflow vessels. Radiographic and anatomical data of the collateral formation in this porcine model has value in investigation of the pathophysiology of hindlimb ischemia, and assessment of angiogenic therapies as potential treatments for PAD.
Abstract 337: Short-Term Protein Restriction Attenuates Vein Graft Disease by Inhibition of Endothelial Cell Damage and Upregulates Cystathionine-g-Lyase
Authors: Kip Peter , Trocha Kaspar M , MacArthur Michael R , Trevino-Villarreal Jose H , Longchamp Alban , de Vries Margreet R , Tao Ming , Mitchell James R , Ozaki C. Keith
Published: March 2019

Objective:
Short-term protein restriction (PR) preceding surgery limits ischemia reperfusion injury and arterial intimal hyperplasia in mouse models, possibly via upregulation of endogenous enzymatic production of the gaseous messenger hydrogen sulfide (H
2
S) by cystathionine-gamma-lyase (CGL). Here, we tested the hypothesis that short-term PR will limit vein graft disease (VGD) via CGL upregulation and increased H
2
S production, resulting in EC cytoprotection and reduced leukocyte transmigration.

Approach and Results:
LDLR
-\-
mice (male, 8-10 weeks old) were fed a high fat/high cholesterol (HF/HC) diet for two weeks. One group was then switched to a PR HF/HC diet with or without daily CGL inhibitor propargylglycine (PAG) injections. 7 days later mice underwent carotid interposition grafting with donor caval veins from mice on matched diets. Mice were euthanized at baseline (preop), post-op day 4 or 28 for analysis of the graft (histology, immunohistochemistry), aorta (western blot) and kidney EC (flow-cytometry).

PR mice showed decreased graft intimal/media+adventitia (I/M+A) area and thickness ratios (Fig. A) on post-op day 28. Staining for CD31 and neutrophil-elastase revealed a relatively intact EC layer (Fig. B-C, CD31 in brown, red arrows, p<0.001) and less neutrophil transmigration (p=0.02) on post-op day 4. PR increased baseline CGL expression in aortas (Fig. D-E) and upregulated EC H
2
S (Fig. F). Our preliminary data indicates that PAG injections during PR tended to diminish its protective effects on VGD (Fig. G).

Conclusions:
PR attenuated VGD via limiting of EC damage concomitant with increased CGL expression and endogenous H
2
S production. Ongoing studies will further define the CGL dependence of this process. Short-term preoperative PR stands as a potentially simple, safe, cost-efficient intervention to enhance vein graft durability.
Abstract 342: Helix-loop-helix Transcription Factor Id3 Promotes Ischemia-induced Angiogenesis
Authors: Osinski Victoria , Ganta Vijay C , Nguyen Anh T , Annex Brian H , McNamara Coleen A
Published: March 2019

The development of new blood vessels is important during the progression of many diseases including cancer, obesity, and ischemia. While inhibiting blood vessel growth can prevent excess adiposity and tumor growth, it can also exacerbate symptoms of ischemia. Using flow cytometry to quantify total endothelial cells (ECs) in the gastrocnemius of mice who have undergone femoral artery resection (FAR), the number of ECs per gram of muscle increases approximately four-fold relative to uninjured gastrocnemius from the same mouse (442% increase, ± 208%) in a model of hindlimb ischemia. The helix-loop-helix (HLH) transcription factor Inhibitor of differentiation (ID3) promotes angiogenesis and whole tissue blood perfusion in models of cancer and obesity, but the potential role for ID3 in promoting ischemia-induced angiogenesis remains uninvestigated. Id3 mRNA levels in injured gastrocnemius are increased three days post-FAR relative to uninjured gastrocnemius (p < 0.05, n = 6). Furthermore, ID3 global knockout (KO) mice have attenuated perfusion recovery compared to wild type littermate controls at 14 days post-FAR (p < 0.05). Gastrocnemius muscles isolated from ID3 global KO mice 7 days post-FAR express higher levels of the cell cycle inhibitor p21
Cip1
mRNA than WT mice (p = 0.10, n = 3). Finally, flow cytometry analysis of cell subsets in a novel Id3 promoter-GFP reporter mouse line revealed that Id3 promoter activation is greater in CD31
+
ECs than many other cell types (n = 10), suggesting ECs may be an important cell type in which ID3 is promoting pro-angiogenic effects. Together, these data support the hypothesis that ID3 promotes ischemia-induced angiogenesis by promoting EC proliferation and inhibiting cell cycle inhibitor p21
Cip1
. To investigate the role of ID3 in ECs during angiogenesis, an EC-specific, tamoxifen-inducible Id3 KO mouse (Id3
fl/fl
Cdh5-Cre/ERT2) has been generated.
Abstract 344: Reconstituted High-density Lipoprotein (rHDL) Directly Modulates Inflammatory Cells after Myocardial Infarction in Mice
Authors: Richart Adele , Reddy Medini , Heywood Sarah , Diditchenko Svetlana , Navdaev Alexei , Kingwell Bronwyn
Published: March 2019

Aim:
We have recently demonstrated that reconstituted high-density lipoprotein (rHDL) delivered immediately after myocardial infarction reduces infarct size and improves heart function in mice (
Heywood SE, Sci Transl Med, 2017
). We now examine potential immunomodulatory actions of HDL that may underlie these effects.

Methods:
In male C57BL/6 mice, a single intravenous bolus of rHDL (CSL-111, 80mg/kg of human apoA-I, or saline) was delivered at the time of reperfusion following 30mins of surgically-induced ischemia. Effects on the inflammatory response were studied throughout the 5 days post ischemia-reperfusion.

Results:
Twenty-four hours after ischemia-reperfusion, rHDL reduced the number of circulating leukocytes (versus saline p<0.05) and increased the number in spleen (p<0.05). rHDL inhibited the recruitment cascade of the entire spectrum of inflammatory cells into the left ventricle (LV) including neutrophils (1 day post ischemia), T and B cells (3 days after), and monocytes (5 days after) (p<0.05 for all). This was associated with lower cardiac expression of chemokines that attract neutrophils (CXCL1, CXCL2, CXCL5) and monocytes (CCL2) (ELISA, p<0.05 for all). Histochemistry at 6 and 24 hours after ischemia-reperfusion showed fluorescently labeled rHDL localized to the infarct and peri-infarct regions. There were also greater quantities of rHDL (human apoA-I) in the LV and spleen of mice subjected to ischemia versus sham-operated mice (ELISA, p<0.05); no rHDL was observed in the right ventricle. In addition, flow cytometry studies using the fluorescent rHDL indicated binding to both circulating leukocytes as well as those recruited into the ischemic LV (mostly neutrophils and monocytes). rHDL had a greater association with the circulating pro-inflammatory monocyte subtype (Ly6C
high
) than the anti-inflammatory subtype (Ly6C
low
; p<0.05) 24 hours post ischemia. Furthermore, there was less recruitment of Ly6C
High
monocytes 5 days post ischemia in the LV of mice treated with rHDL (p<0.05).

Conclusion:
rHDL limits the myocardial post-ischemic inflammatory response by effects in both the LV and by directly modulating inflammatory cells. These finding suggest a novel treatment modality for acute coronary syndromes.
Abstract 346: Pre-Operative Protein and Methionine Restriction Does Not Impair Wound Healing in a Murine Mcfarlane Wound Healing Model
Authors: Trocha Kaspar
Published: March 2019

Background:
Numerous pre-clinical studies have demonstrated that pre-conditioning before surgery with various forms of dietary restriction enhances the surgical stress response leading to improved outcomes. However the effect of restricting proteins or amino acids for seven days prior to surgery on a healing surgical wound is currently unknown. Therefore using a using an established murine wound healing model we hypothesized that pre-operative protein restriction (PR) or methionine restriction (MR) would not impair wound healing.

Approach & Results:
To asses wound healing potential a Mcfarlane wound healing model was employed where a pedicle flap measuring 2.5 x 1.25cm with a silicone sheet inferior to flap to prevent angiogenesis from bellow on the dorsum of the mouse was performed. Dietary interventions included 7 days of either an iso-caloric protein restricted (90% carb., 10% fat, 0% protein) diet or 3 weeks of an methionine restricted (76% carb., 10% fat, 14% protein) diet before surgery and resumed a complete (72% carb., 10% fat, 18% protein) diet post op. Daily photographs of the wound were taken and necrotic area was calculated. PR mice revealed a trend towards accelerated wound healing, with no difference in percentage of viable skin 7 days after surgery (Fig. A). Also, daily laser Doppler imaging was performed to assess for flap perfusion and viability (Fig. B,E). MR mice displayed accelerated wound healing 3 days after surgery (Fig. C, p<0.05) compared to controls while after 7 days, as with PR mice, no difference in percentage viable skin was observed (Fig. D).

Conclusion:
Reducing/removing protein intake or eliminating single amino acids prior to surgery in pre-clinical models has shown to protect against surgical stress. Here we show that brief dietary-manipulations stand as simple strategies toward improving the host response to surgical injury and do not inhibit or impair wound healing in mice, thereby further enhancing clinical applicability.
Abstract 350: Brown Adipose Tissue, a Novel Mechanism to Induce both Angiogenesis and Arteriogenesis
Authors: Zhang Jie , Oydanich Marko , Vatner Dorothy E , Vatner Stephen F
Published: March 2019

Brown adipose tissue (BAT) is known to protect metabolism, but the extent to which it mediates angiogenesis and cardioprotection, the goal of this investigation, is not known. The Regulator of G Protein Signaling 14 (RGS14) knockout (KO) mouse is unique in that it not only extends longevity, but also enhances several aspects of healthful aging, i.e., protects against diabetes, obesity, exercise intolerance and increased brown adipose tissue by 47% compared with wild type littermates (WT). However, less is known about its role in cardioprotection. Accordingly we examined the effects of chronic myocardial ischemia after 1 and 3 weeks of permanent coronary artery occlusion (CAO) in 3-4 month old RGS14 KO and their WT. Scar size after 3 weeks CAO, determined by histology, was decreased by 33±5% in RGS14 KO, which was only possible in the face of permanent CAO if angiogenesis/arteriogenesis developed in the RGS14 KO mice hearts. Indeed, both angiogenesis assessed by Ki 67 endothelial cell staining and arteriogenesis, assessed by Ki 67 arteriole staining, more than doubled compared with WT. This was associated with a 206±25.5% increase in VEGF in the RGS14 KO hearts compared with WT. We then examined the role of BAT by using a simulated BAT KO, i.e., BAT was transplanted from the RGS14 KO to WT. In the WT with BAT transplants VEGF was increased by 90%. Infarct size, assessed as the fraction of area at risk, was lower, p<0.05, in the BAT transplants (27±2.8%), compared with infarct size in RGS14 KO donors (44±1.9%), a value similar to that observed in WT without the BAT transplant (46±1.0%). Thus, brown adipose tissue appears to be a novel mechanism to induce both angiogenesis and arteriogenesis, resulting in vascular protection from ischemia.
Abstract 353: Aldehyde Scavenging Reduces Renal Injury Role of IsoLG-modified apoAI
Authors: Kon Valentina
Published: March 2019

Reactive aldehydes, including isolevuglandins (IsoLG) are lipoxidation products that can modify lipoproteins and promote cellular dysfunction. Previously, we showed that proteinuric injury increases urinary apoAI and causes renal lymphangiogenesis. We now investigate the role of IsoLG and effects of IsoLG scavenging on renal injury and lymphatics.

Nphs1-hCD25 mice (NEP25) expressing podocyte-specific human CD25 become proteinuric after injection of immunotoxin (LMB2). NEP25 mice were treated with the aldehyde scavenger, PPM (1g/L) or vehicle from the onset of proteinuria until sacrifice (2 weeks). We assessed proteinuria [measured as albumin:creatinine ratio (ACR)], urinary apoAI, IsoLG, KIM-1 (marker of tubular injury), and the renal expression of lymphatic markers (LYVE-1 and podoplanin).
In vitro
, we assessed the effects of apoAI or modified apoAI (IsoLG-apoAI) +/- PPM on proximal tubular cells (PTC) and lymphatic endothelial cells (LEC).

As expected, LMB2 significantly increased ACR and urinary KIM-1. Proteinuric injury also significantly increased urinary IsoLG (262%). Similar to our previous data, proteinuric kidneys increased expression of apoAI, VEGF-C, podoplanin and LYVE-1

compared to wild type mice.
In vitro
, PTC uptake of IsoLG-apoAI was significantly increased (39%), an effect that was abrogated by exposure to PPM (22%). In LECs, compared to apoAI, IsoLG-apoAI significantly increased viability (27%) and migration (13%), effects that were abrogated by exposure to PPM.
In vivo
, proteinuric NEP25 mice treated with PPM showed significantly reduced ACR (22%), urinary KIM-1 (30%) and IsoLG excretion (64%).

We conclude that aldehyde scavenger lessens proteinuric renal damage through mechanisms that include reducing proximal tubule uptake of IsoLG-modified apoAI and preserving lymphatic endothelial cell functionality.
Abstract 354: Early Intentional Restoration of Blood Flow Reduces Thrombus Burden and Vein Wall Scarring Following Dvt: Implications for Preventing the Post-thrombotic Syndrome
Authors: Li Wenzhu , Kessinger Chase , Orii Makoto , Kellnberger Stephan , Cui Jie , Mauskapf Adam , Wang Lang , Zheng Xiaoxin , Henke Peter , Jaffer Farouc
Published: March 2019

Background:
Despite anticoagulation therapy, up to 50% of deep vein thrombosis (DVT) patients still develop the post-thrombotic syndrome (PTS), a condition that arises from thrombus obstruction and vein wall damage, leading to venous hypertension. While catheter-based intervention may reduce PTS, the CaVenT and ATTRACT trials demonstrated little clinical benefit of intentional restoration of blood flow (RBF) for reducing PTS. However, these trials have not explored the time-dependence of RBF, a key factor as aging VT are associated with greater vein wall injury.

Methods:
To investigate the temporal effects of intentional RBF following VT, we modified a classic complete ligation inferior vena cava (IVC) stasis VT protocol in C57BL/6 mice by de-ligating the day 0 ligature at day 2 to allow RBF. Compared to intravital microscopy, the sensitivity and specificity of noninvasive ultrasound (US) to detect RBF was 82.1% and 94.4%, respectively. Serial US was then performed on mice. Mice were classified as early RBF (RBF by day 4), late RBF (RBF after day 4), no RBF, or sham de-ligation groups. Follow day 8 sacrifice, thrombus burden (mg/cm) and vein wall scarring (VWS) were measured. Kruskal-Wallis followed by Dunn’s test was used to compare statistical significance between groups.

Results:
After de-ligation, RBF through VT increased gradually over time (Fig. A). The early RBF group exhibited significant reductions in VT burden (N=9) and VWS (N=5) at day 8 (
p
<0.05 vs. comparator groups, Fig B-C). In contrast, the late RBF group did not experience significant reductions in thrombus burden (N=6) nor VWS (N=4), with measures similar to mice with no RBF (N=3-4), or sham de-ligation (N=8-9).

Conclusions:
Intentional early RBF reduces venous thrombus burden and VWS, factors that drive PTS. Mechanistic studies to address these findings are going. The overall results suggest that anti-PTS benefits of catheter-based intervention may be enhanced in subjects receiving early RBF.
Abstract 360: Rivaroxaban Enhances the Antiplatelet Activity of Aspirin via Inhibition of Nox2-mediated Thromboxane A
2
and Isoprostane Biosynthesis
Authors: Cammisotto Vittoria , Carnevale Roberto , Nocella Cristina , Stefanini Lucia , Pastori Daniele , Bartimoccia Simona , Pignatelli Pasquale , Violi Francesco
Published: March 2019

Background:
A recent study demonstrated that a combination of rivaroxaban, an inhibitor of factor Xa, plus aspirin (100mg/day) reduces cardiovascular events in patients with stable vascular disease, compared to aspirin alone but the underlying mechanism is still unknown.

Methods:
In vitro study was performed consisting in measuring agonist-induced platelet aggregation, thromboxane (Tx)B
2
and isoprostane biosynthesis, soluble Nox
2
-dp, a marker of Nox2 activation, and PLA
2
activation in platelets from healthy subjects (n=5) added with or without scalar doses of aspirin (25-100 uM) and/or rivaroxaban (15-120 ng/ml). Furthermore, we measured urinary excretion of isoprostanes, and soluble Nox2 before and after 3 months of 20 mg rivaroxaban (n=25) or warfarin (n=25) treatment in atrial fibrillation(AF) patients.

Results:
In vitro study showed that rivaroxaban significantly reduced platelet aggregation, TxB
2
and isoprostane biosynthesis and Nox2 and PLA
2
activation in a dose-dependent fashion. Also, rivaroxaban dose-dependently inhibited aggregation of platelets treated with low-(25uM) as well as high-(100uM) aspirin doses. In low-dose aspirin-treated samples an incremental inhibition of platelet TxB
2
biosynthesis was achieved; conversely, in platelets treated with high doses of aspirin a significant inhibition of isoprostanes and Nox2 was observed. Inhibition of platelet aggregation by rivaroxaban was detected also in platelets stimulated with convulxin suggesting that its antiplatelet effect was GPVI-dependent. In addition, convulxin elicited PLA
2
activation, that was inhibited by rivaroxaban as well as by a Nox2 inhibitor. Finally, in AF patients treated with rivaroxaban a significant reduction of urinary isoprostanes and soluble Nox2 and GPVI as compared to warfarin-treated ones was detected.

Conclusion:
Here we show that rivaroxaban inhibits platelet aggregation via GPVI interaction and eventually Nox2-mediated thromboxane and isoprostane biosynthesis in aspirin-treated platelets so providing a novel mechanistic insight into the beneficial effects of combining rivaroxaban with aspirin.
Abstract 361: Multifocal Vascular Disorders Due to the Uncommon Anti-phosholipid Antibody Syndrome
Authors: Kawanura Yasutaka , Ai Masato , Nakaki Masafumi
Published: March 2019

Purpose:
We report three cases with uncommon anti-phosholipid antibody syndrome. The difficulty in diagnosing as well as treatment procedures for multifocal vascular disorders is discussed.

Patients and Methods:
All patients were examined and were initially treated for digestive disorders.

Patient 1, 84-year-old woman, with gastrointestinal bleeding

Patient 2, 76-year-old man, with sudden onset of upper abdominal pain

Patient 3, 54-year-old man, with nausea and vomiting, liver dysfunctionClinical manifestation was superior mesenteric artery (SMA) thrombosis in the patient 1 and 2, and portal vein thrombosis in the patient 3.

Serial CT scans were examined on 64 multidetector-row CT; Hitachi and GE, with bolus injection of contrast media for CT angiography.

Abdominal angiography and coronary angiography with endovascular intervention performed on were performed on Allura; Philips.

Results:
CTA of for patient 1 and 2 showed decreased contrast enhancement of SMA and SMV enhancement. The intestinal enhancement was poor in patient 1 and well preserved in patient 2. A week later, patient 1 died suddenly due to multiple organ failure. Although the symptom improved in patient 2, the follow-up CT revealed the enlargement ofSMA thrombosis. The aspiration and the dissolution of SMA thrombosis were performed and resulted in complete recanalization. Then he felt chest oppression and 75% stenosis of RCA #4, followed by complete spastic occlusion on Acetylcholine loading on the coronary angiography, which recovered perfectly with intracoronary nitroglycerin administration.He discharged our hospital with health, while he developed cerebellar infarction. On patient 3, to resolve portal hypertension, balloon-occluded retrograde transvenous obliteration and splenic arterial embolization were successfully performed.

Discussion and Conclusions:
Acute deterioration encountered in patient 1, the progressive SMA thrombosis with coronary disease and subsequent cerebellar occlusion seen in patient 2, and portal vein thrombosis and portal hypertension without chronic hepatitis, cirrhosis or alcoholic liver damage in the patient 3 were seemed to be unusual. To discover underlining disorders such as uncommon anti-phosholipid antibody syndrome is important to prevent serious vascular diseases in sequence.
Abstract 378: Predilection of Low Protein C-induced Spontaneous Atherothrombosis for the Right Coronary Sinus in Apolipoprotein E-deficient mice
Authors: Heestermans Marco , Ouweneel Amber B , Maracle Chrissta X , Hassan Jasmin , Kloosterman Meander , Reitsma Pieter H , Gijbels Marion J , van Vlijmen Bart J , Van Eck Miranda , Van Eck Miranda
Published: March 2019

Atherothrombosis is the cause of death of over 14 million people per year worldwide and murine models to replicate this process
in vivo
are mostly lacking. Previously we demonstrated that silencing of anticoagulant protein C using RNA interference (
siProc
) induces spontaneous atherothrombosis in the aortic root of apolipoprotein E-deficient (
Apoe
-/-
) mice, albeit at a low incidence rate. Here we aim to determine if plaque susceptibility for rupture can be linked to plaque characteristics and/or blood composition, and moreover, we attempt to boost incidence through a transient increase in blood pressure as well as to localize atherothrombosis to an additional predefined vascular site by means of a semi-constrictive collar around the carotid artery. In the current study, si
Proc
-driven spontaneous atherothrombosis in the aortic root of Apoe-/- mice was reproduced and occurred at an incidence of 23% (9 out of 39 mice), while the incidence of collar-induced atherothrombosis in the carotid artery was 2.6% (1 out of 39 mice). Treatment with phenylephrine, to transiently increase blood pressure, did not increase atherothrombosis in the aortic root of the
Apoe
-/-
mice nor in the carotid arteries with collars. Plaques in the aortic root with an associated thrombus were lower in collagen and macrophage content, and mice with atherothrombosis had significantly more circulating platelets. Plasma protein C, white blood cell counts, total cholesterol, fibrinogen, and serum amyloid A were not different amongst si
Proc
-treated mice with or without thrombosis. Remarkably, our data revealed that thrombus formation preferably occurred on plaques in the right coronary sinus of the aortic root. In conclusion, there is a predilection of low protein C-induced spontaneous atherothrombosis in
Apoe
-/-
mice for the right coronary sinus, a process that is associated with an increase in platelets and plaques lower in collagen and macrophage content.
Abstract 379: Platelet-driven Contraction of Venous Thrombi Modulates Their Obstructiveness and Embologenicity
Authors: Tutwiler Valerie , Peshkova Alina D , Le Minh Giang , Andrianova Izabella A , Weisel John W , Litvinov Rustem I
Published: March 2019

Contraction (retraction) of the blood clot is a part of the clotting process driven by activated platelets attached to fibrin. The aim of this work was to reveal the pathogenic importance of contraction of clots and thrombi in venous thromboembolism (VTE). We investigated the kinetics of clot contraction in clots made from the blood of 55 patients with VTE not receiving antiplatelet and anticoagulant medications. In addition, we studied the ultrastructure of
ex vivo
venous thrombi, as well as the morphology and functionality of isolated platelets. Thrombi from VTE patients contained compressed polyhedral erythrocytes, a marker for clot contraction
in vivo
. The extent and rate of contraction of in vitro clots were reduced by 2-fold in clots from the blood of VTE patients compared to healthy controls. The contraction of clots from the blood of patients with pulmonary embolism was significantly impaired compared to that of those with isolated venous thrombosis, suggesting that less compacted thrombi may be prone to embolization. The reduced ability of clots to contract correlated with continuous platelet activation followed by their partial refractoriness to stimulation. Morphologically, 75% of platelets from VTE patients were spontaneously partially activated (with filopodia) compared to only 21% of those from healthy controls. At the same time, platelets from VTE patients showed a 1.4-fold reduction in activation markers expressed in response to chemical activation when compared to platelets from healthy individuals. The results obtained suggest that the impaired contraction of thrombi resulting from platelet dysfunction is an underappreciated pathogenic mechanism in VTE that may regulate the obstructiveness and embologenicity of venous thrombi. Furthermore, an assay for clot contraction may have diagnostic and prognostic value for venous thromboembolism.

The work was supported by the Program for Competitive Growth at Kazan Federal University.
Abstract 383: HDL Particles, Cell-Cholesterol Efflux, and CHD Risk: the Preβ-1 Paradox
Authors: Asztalos Bela F , Horvath Katalin V , Schaefer Ernst J
Published: March 2019

Objective:
The cell-cholesterol efflux capacity (CEC) of high-density lipoprotein (HDL) is inversely associated with coronary heart disease (CHD) risk. ATP-binding cassette transporter-A1 (ABCA1) plays a crucial role in cholesterol efflux from macrophages to preβ-1-HDL. We tested the hypothesis that CHD patients have functionally abnormal preβ-1-HDL.

Approach and Results:
We measured HDL CEC via the ABCA1 and the scavenger receptor class B type-I (SR-BI) pathways, HDL anti-oxidative capacity, apolipoprotein A-I-containing HDL particles, and inflammatory- and oxidative-stress markers in 64 CHD cases and 64 gender-matched controls. There were significant positive correlations between ABCA1-dependent cholesterol efflux and the levels of small lipid-poor preβ-1-particles in both cases (R
2
=0.541) and controls (R
2
=0.350). Cases had almost twice the levels of preβ-1-HDL than controls, but the functionality of their preβ-1 particles (preβ-1-concentration-normalized ABCA1-dependent efflux capacity) was significantly lower (-39%). There were significant positive correlations between SR-BI-dependent cholesterol efflux and the levels of large lipid-rich (α-1 + α-2) HDL particles in both cases (R
2
=0.828) and controls (R
2
=0.671). Cases had significantly lower (-11%) levels of large HDL particles, but the functionality of their particles (large-HDL-concentration-normalized SR-BI-dependent efflux capacity) was significantly higher (+27%) compared to controls. High plasma levels of inflammatory and oxidative stress markers were not associated with the functionality of HDL particles. High plasma levels of triglycerides were associated with increased HDL particle functionality. HDL anti-oxidative capacity was significantly lower (-15%) in cases than in controls. There were no significant correlations between HDL anti-oxidative capacity and the concentrations of inflammatory- and oxidative-stress markers or HDL CEC.

Conclusions:
HDL CEC is significantly influenced by both the concentration and the functionality of specific HDL particles. CHD patients have higher than normal preβ-1 concentration with decreased functionality and lower than normal large-HDL particle concentration with enhanced functionality.