bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 255: Fatty Acid Binding Protein 4, FABP4, Causes Impaired Wound Healing in Diabetes
Authors: Boniakowski Anna , Kimball Andrew , Davis Frank , Joshi Amrita , Schaller Matt , denDekker Aaron , Kunkel Steve , Gallagher Katherine
Published: March 2019

Wound healing in diabetes is impaired due to failed resolution of inflammation. Macrophages play a significant role in the establishment of a regulated inflammatory response during wounding. Macrophage function is dictated by metabolism, which alters gene expression. Recent studies suggest that a fatty acid binding protein, FABP4, may control macrophage function in diabetes by altering metabolism.

Thus, we examined whether FABP4 controls macrophage function and hence inflammation in diabetic wound healing. To investigate this, C57BL/6 mice were fed either a normal (12% saturated fat) diet or a high-fat (60% saturated fat) diet (HFD) for 12 weeks to induce physiologic “pre-diabetes.”

Wounds were created and CD3-CD19-NK1.1-CD11b+ cells (macrophages) were isolated each day following injury and FABP4 expression was quantified by qPCR and Western blot. We found that HFD wound macrophages demonstrated a significant increase in FABP4 gene expression and protein production on day 3 post-injury compared with controls.

To determine if FABP4 alters inflammatory gene expression in wound macrophages, we isolated wound macrophages with an FABP4 inhibitor, treated them, and analyzed for IL-1β and TNFα expression.

IL1β and TNFα gene expression were significantly reduced (P<0.01) in diabetic wound macrophages treated with the FABP4 inhibitor, suggesting that inflammatory gene expression can be controlled in diabetic wound macrophages through FABP4 modulation. As we have previously identified, epigenetic mechanisms often dictate gene expression during wound healing, thus we examined whether FABP4 expression in diabetic macrophages was regulated by histone modifications.

Chromatin immunoprecipitation (ChIP) analysis of the FABP4 promoter in wound macrophages revealed a significant increase in H3K4 trimethylation, an activating mark, on the FABP4 promoter in diabetic wound macrophages suggesting that epigenetic regulation may play an important role in the differential expression of FABP4 in diabetic wounds.

In conclusion, FABP4 appears to be upregulated in diabetic wound macrophages and contributes to increased macrophage inflammation. Modulation of FABP4 or its expression may help resolve inflammation in diabetic wounds and promote healing.
Abstract 273: Cell Mimetic Liposomal Nanocarriers Tailored for Vascular Smooth Muscle Cell Molecular Therapeutics
Authors: Mattern-Schain Samuel I , Fisher Richard K , Grimsley Lauren B , Kirkpatrick Stacy S , Grandas Oscar H , Best Michael D , Mountain Deidra J
Published: March 2019

Introduction:
Our laboratory aims to develop biocompatible nanocarriers for molecular therapeutics aimed at vascular pathology. We have previously established a liposome platform that is an effective delivery system for RNAi in vascular smooth muscle cells (VSMC). Tailoring liposome membranes to mimic vascular cell membrane lipid constituents may be a promising strategy for increased delivery to target cells. Here we test our previously established liposome platform with the incorporation of naturally occurring signaling lipids known to influence vascular cell function as a method to increase VSMC association.

Methods:
Established cell-penetrating neutral liposomes (R8-PLPs) were assembled and fluorescently tagged as previously described. The propensity of diacylglycerol (DAG) and/or phosphatidylserine (PS) to increase the association of R8-PLP to VSMCs was tested by the incorporation of gradient percentages DAG/PS alone and in combination at 5-20% membrane occupancy. Liposome stability and siRNA encapsulate retention was analyzed via dynamic light scattering and Ribo-green, respectively.

Results:
DAG and PS incorporation increased VSMC association of R8-PLP, with 10% PS increased over all other groups (P10; Fig1A). Combinatorial formulations were screened for optimal DAG content with PS fixed at 10%. DAG20%+PS10% (D20P10) performed best, with increased VSMC association over all other combinatorial groups or independent P10 modification (Fig1B). Stability profiles were consistent (~50nm size and ~80% drug retention) and not significantly different among groups.

Conclusion:
Signaling lipid incorporation into the nanocarrier architecture potentiates VSMC association of established R8-PLP liposomes, without sacrificing stability or drug retention. These results suggest cell mimetic tuning of liposomes to generate specificity and increase delivery efficacy is a viable strategy for advancing targeted liposomal drug delivery.
Abstract 306: Endothelial 6-Phosphofructo-2-Kinase/Fructose-2,6-Bisphosphatase, Isoform 3 (PFKFB3) Deficiency Inhibits Hypoxia-Induced Pulmonary Hypertension in Mice
Authors: Cao Yapeng , Yang Qiuhua , Wang Lina , Xu Jiean , Liu Zhiping , Xu Yiming , Wang Yong , Fulton David J , Weintraub Neal L , Wu Chaodong , Li Qinkai , Su Yunchao , Hong Mei , Huo Yuqing
Published: March 2019

Background:
Pulmonary artery hypertension (PAH) is a severe proliferative disease characterized by the remodeling of small pulmonary arteries leading to a progressive increase in pulmonary vascular resistance and ultimately to right ventricular failure and death. Many studies have indicated that endothelial dysfunction is a key element in the pathogenesis of this disease. In this study, we investigated whether endothelial glycolysis plays a critical role in the development of hypoxia-induced PAH by knocking down endothelial PFKFB3 (encoding 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, isoform 3), a critical regulator of glycolysis.

Methods and Results:
Immunostaining results showed that pulmonary artery endothelial PFKFB3 was increased in the lung of PAH patients. Mice deficient in endothelial
Pfkfb3
showed decreased right ventricular systolic pressure, attenuated right ventricular hypertrophy and normal morphology of distal pulmonary arteries after four weeks of hypoxia compared with control mice. Furthermore, PFKFB3-deficient pulmonary arterial ECs secreted less growth factors, which ameliorated the proliferation of pulmonary artery smooth muscle cells. Additionally, the levels of endothelial derived inflammatory cytokines were also decreased. Besides, the inhibitory effect of endothelial
Pfkfb3
supression on PAH formation were demonstrated in Sugen 5416/hypoxia rat pulmonary hypertension model with 3PO, a specific PFKFB3 inhibitor. Mechanistically, genetic deletion of
PFKFB3
or 3PO treatment decreased the TNF-α induced NLRP3 inflammasome activation in pulmonary arterial ECs.

Conclusions:
Endothelial PFKFB3 inhibition is able to ameliorate the development of hypoxia-induced pulmonary hypertension.
Abstract 315: SmgGDS Prevents Thoracic Aortic Aneurysm Formation and Rupture by Phenotypic Preservation of Aortic Smooth Muscle Cells
Authors: Nogi Masamichi , Satoh Kimio , Sunamura Shinichiro , Kikuchi Nobuhiro , Satoh Taijyu , Kurosawa Ryo , Omura Junichi , Al-Mamun MD Elias , Siddique Mohammad Abdul Hai , Kudo Shun , Miyata Satoshi , Shimokawa Hiroaki
Published: March 2019

Background:
Thoracic aortic aneurysm (TAA) and dissection (TAD) are fatal diseases, which cause aortic rupture and sudden death. The small GTP-binding protein GDP dissociation stimulator (SmgGDS) is a crucial mediator of the pleiotropic effects of statins. Previous studies revealed that reduced force generation in AoSMCs causes TAA and TAD.

Methods and Results:
To examine the role of SmgGDS in TAA formation, we employed an angiotensin II (AngII, 1,000 ng/min/kg, 4weeks)-induced TAA model in
Apoe

-/-
SmgGDS

+/-

mice, in which 33% died suddenly due to TAA rupture, whereas there was no TAA rupture in
Apoe

-/-

control mice. In contrast, there was no significant difference in the ratio of AAA rupture between the two genotypes. We performed ultrasound imaging every week to follow the serial changes in aortic diameters. The diameter of the ascending aorta progressively increased in
Apoe

-/-
SmgGDS

+/-

mice compared with
Apoe

-/-

mice, whereas that of the abdominal aorta remained comparable between the two genotypes. Histological analysis of
Apoe

-/-
SmgGDS

+/-

mice showed dissections of major thoracic aorta in the early phase of AngII infusion (day 3~5) and more severe elastin degradation compared with
Apoe
-/-
mice. Mechanistically,
Apoe

-/-
SmgGDS

+/-

mice showed significantly higher levels of oxidative stress, matrix metalloproteinases, and inflammatory cell migration in the ascending aorta compared with
Apoe

-/-

mice. For mechanistic analyses, we primary cultured AoSMCs from the 2 genotypes. After AngII (100 nM) treatment for 24 hours,
Apoe

-/-
SmgGDS

+/-

AoSMCs showed significantly increased MMP activity and oxidative stress levels compared with Apoe
-/-
AoSMCs. In addition, SmgGDS deficiency increased cytokines/chemokines and growth factors in AoSMCs. Moreover, expressions of
FBN1
,
ACTA2
,
MYH11
,
MLK
and
PRKG1
, which are force generation genes, were significantly reduced in
Apoe

-/-
SmgGDS

+/-

AoSMCs compared with Apoe
-/-
AoSMCs. Similar tendency was noted in AoSMCs from TAA patients compared with those from controls. Finally, local delivery of the SmgGDS gene construct reversed the dilation of the ascending aorta in
Apoe

-/-
SmgGDS

+/-

mice.

Conclusions:
These results suggest that SmgGDS is a novel therapeutic target for the prevention and treatment of TAA.
Abstract 332: Fibronectin Containing Extra Domain a in the Plasma, but not Tissue, Exacerbates Stroke Outcome in the Comorbid Condition of Hyperlipidemia
Authors: Dhanesha Nirav , Chorawala Mehul , Doddapattar Prakash , Chauhan Anil
Published: March 2019

Background:
Fibronectin-splicing variant containing extra domain A (Fn-EDA) is expressed in the endothelium of atherosclerotic arteries and elevated in circulation in at risk of patients with diabetes, hypertension, and atherosclerosis. Recent studies suggest that Fn-EDA contributes to ischemic stroke. The relative contribution of plasma versus (vs.) tissue Fn-EDA in stroke outcome remains unclear. We determined the contribution of plasma vs. tissue Fn- EDA in stroke exacerbation in the comorbid condition of hyperlipidemia.

Methods:
We generated
Fn-EDA

fl/fl
AlbCre

+

, which express Fn-EDA in all tissues but not in plasma, on hyperlipidemic apolipoprotein E deficient (
Apoe

-/-

) background. Controls were
Fn-
EDA

fl/fl
Apoe

-/-

mice, which express FN-EDA in all tissues and plasma. Susceptibility to stroke outcome was evaluated in male and female mice (8-10 weeks) by transient 1-hour ischemia followed by 1, 3, and 7 days of reperfusion. Quantitative assessment of stroke outcome was evaluated by measuring infarct volume (MRI), cerebral blood flow (laser speckle imaging), neurological outcome, and postischemic thrombo-inflammation (fibrin deposition, neutrophil, and inflammatory cytokines within the infarcted and surrounding region by Western blotting and immunohistochemistry). Susceptibility to thrombosis was evaluated in a FeCl
3
injury- induced carotid thrombosis model.

Results:
Irrespective of gender,
Fn-EDA

fl/fl
AlbCre

+
Apoe

-/-

exhibited smaller infarcts and improved neurological outcome at days 1, 3 and 7 concomitant with improved survival, and decreased post ischemic thrombo-inflammation (
P
<0.05 vs.
Fn-EDA

fl/fl
Apoe

-/-

). Laser speckle imaging revealed improved regional cerebral blood flow in
Fn- DA

fl/fl
AlbCre

+
Apoe

-/-

mice (
P
<0.05 vs.
Fn-EDA

fl/fl
Apoe

-/-

), suggesting that Fn-EDA present in plasma may promote intracerebral thrombosis and thereby exacerbate stroke. Using i ntravital microscopy, we found that
Fn-EDA

fl/fl
AlbCre

+
Apoe

-/-

mice were less susceptible to experimental thrombosis (
P
<0.05 vs.
Fn-EDA

fl/fl
Apoe

-/-

).

Conclusion:
Following STAIR guidelines, we provide evidence that Fn-EDA present in the plasma, but not tissue, contribute to stroke exacerbation by promoting thrombo-inflammation.
Abstract 336: MiR-126 Modified Endothelial Progenitor Cells Transplantation Contributes to Angiogenesis after Brain Focal Ischemia in SHR Rats
Authors: Huang Jun , Qu Meijie , Song Yaying , Lu Yuanyuan , Gao Pingjin
Published: March 2019

Background and Purpose:
Transplantation of endothelial progenitor cells (EPCs) leads to better outcomes in experimental stroke, while improve the EPCs survival rate in ischemia area is still a challenge. MiR-126 modulates vascular development and angiogenesis. Here we overexpressed miR-126 in transplanted EPCs, to investigate the function of gene modified EPCs in angiogenesis after brain ischemia.

Methods:
Adult male SHR rats underwent permanent suture middle cerebral artery occlusion (MCAO). One week after middle cerebral artery occlusion, the animals received tail vein injection of miR-126 modified EPCs as treatment or EPCs as control and were monitored for 5 weeks. Brain water content, infarct volume, neurological score, neurogenesis and angiogenesis were examined.

Results:
Neurological score was greatly improved and brain atrophy was greatly reduced in miR-126 modified EPCs-treated SHR rats compared with the control rats 5 weeks after MCAO (P<0.05). The number of bromodeoxyuridine+/CD31+ microvessels are significantly increased. EPCs migration and proliferation were promoted after miR-126 modified in vitro.

Conclusions:
Our results showed that miR-126 modified EPCs therapyreduced ischemic brain injury, along with increased angiogenesis and neurogenesis in SHR rats, suggesting miR-126 significantly improved EPCs function in angiogenesis after MCAO. Gene modified EPCs represents a promising avenue for ischemic stroke stem cell therapy.
Abstract 339: Predictors of Periopertive Neurologic Outcomes Following CEA
Authors: Macharzina Roland R , Vogt Matthias , Mueller Carolin , Neumann Franz-Josef , Zeller Thomas
Published: March 2019

Objective:
The prospective registry study intended to identify risk factors of periinterventional neurologic outcomes after carotid endarteriectomy (CEA) to improve patient selection. Neurologic outcome predicition differs from the endpoint MACCE as previously shown.

Methods:
Patient characteristics and procedural factors were prospectively acquired and analysed using Cox regression to determine predictors for stroke and death within 30-days of CEA.

Results:
The analysis included 748 operations, 262 (35%) asymptomatic, 208 (28%) with previous strokes, and 278 (37%) with transient ischemic attacks (TIA). The overall 30-day stroke rate was 5.5%, 3.1% in asymptomatic and 6.8% in symptomatic patients and for stroke and death 5.6%, 3.1% and 7.0% respectively. Independent predictors of stroke and death were contralateral occlusion (HR=3.826, p=0.006, CI 1.483 - 9.867), symptomatic status (HR=3.626, p=0.004, CI 1.51 - 8.706), First-degree atrioventricular block (HR=2.209, p=0.041, CI 1.034 - 4.722), resection (HR=2.448, p=0.027, CI 1.106 - 5.416) and myocardial infarction (HR=2.03, p=0.04, CI 1.032 - 3.994) compared to all patients. Similar effect estimates were found for stroke alone. Compared to lower risk patients, SHR and SEC criteria predicted stroke and death after adjustment for symptomatic status (HR=2.29, p=0.02, CI 1.142 - 4.592) and (HR=2.286, p=0.028, CI 1.094 - 4.778). Outcome in low risk patients were not related to the symptomatic status for stroke and death (HR=2.484, p=0.148, CI 0.724 - 8.524), correspondingly for stroke.

Conclusions:
This is the first report identifying preprocedural myocardial infarction, first degree AV-block, SHR and SEC as predictors of stroke and stroke and death at 30 days postoperatively.
Abstract 351: National Rates of Venous Thromboembolism in Patient with STEMI: Results from the National Inpatient Sample 2003-2013
Authors: Ayoub Ali , Al-Ogaili Ahmed , Fuentes Harry E , Sleiman Jose , Torres Christian , Diaz Quintero Luis , Tafur Alfonso
Published: March 2019

Background:
There is a growing body of evidence against the use of triple therapy (anticoagulation and dual antiplatelet) among patients with acute coronary syndrome. Venous thromboembolism (VTE) is another potential indication for triple therapy, but the magnitude of such problem is unknown. Therefore, we aimed to determine the trends of annual rate of immediate VTE occurrence in patients with ST-segment elevation myocardial infarction (STEMI) and measure its impact.

Methods:
We queried the 2003-2013 Nationwide Inpatient Sample to identify adults with primary diagnosis of STEMI using ICD-9-CM codes. VTE, including limb vein thrombosis and pulmonary embolism, was allocated when present among the secondary discharge diagnosis. Demographics and inpatient outcomes were compared in the VTE and non-VTE group.

Results:
From 2,495,757 hospitalizations for STEMI, 15,471 (0.6%) also experienced VTE. The group who experienced VTE was older (mean age: 68.44 vs 64.81, p<0.01) and had higher proportions of black patients (10.6% vs 7.7%, p<0.001) and females (42.1% vs 35%, p<0.001) compared to the non-VTE group. There was an increasing trend in the annual rate of VTE during the study period (2003: 0.3% vs 2013: 0.9%, p < 0.01). Patients with VTE had a prolonged hospitalization (12.8 vs 4.63 days, p <0.01), higher risk of gastrointestinal bleeding (OR:1.65; 95% CI 1.54-1.76, p<0.01) and intracranial hemorrhage(OR:1.52, 95% CI:1.23-1.87, P<0.01), needed more blood transfusions (OR:1.74; 95% CI:1.66-1.82, p<0.01) and had increased mortality (OR:1.53, 95%CI:1.46-1.6, p<0.01).

Conclusion:
There is an increasing annual rate in immediate VTE occurrence in patients with STEMI. VTE is associated with more bleeding complications, longer hospital stay, and higher mortality. It is plausible that individualized or more aggressive protocols of VTE prophylaxis are needed in this population.
Abstract 352: National Trends in Cancer-Associated Thrombosis Hospitalizations among Patients with Pancreatic, Gastric and Lung Primary: Results from Nationwide Inpatient Sample Database 2009-2014
Authors: Fuentes Harry E , Al-Ogaili Ahmed , Ayoub Ali , Paz Luis H , Oramas Diana M , Diaz Quintero Luis , Tafur Alfonso
Published: March 2019

Introduction:
Venous thromboembolism (
VTE
) is a leading cause of morbidity and mortality in cancer; and its occurrence leads to an increased number of hospitalizations. Therefore, we aimed to determine the trends in the annual rate of cancer-associated thrombosis (
CAT
) hospitalizations and measure its impact in patients with highly thrombogenic tumor types, such as pancreas, stomach and lung.

Methods:
We queried the 2009-2014 Nationwide Inpatient Sample database to identify adults with gastric, pancreatic or lung cancer. Hospitalizations in which VTE was among the top-three discharge diagnoses, were considered as CAT admissions. In-hospital outcomes of patients with VTE were compared to those without VTE. Using SPSS, version 24, we conducted a linear regression analysis for trend and binary logistic regression analysis to obtain adjusted odds ratios (
OR
)

Results:
From 3 million admissions, 149.577 (4.9%) were related to VTE. The patients were mainly white (68.9%) men (53.2%) with a median age of 69 (IQR:17) years; and the predominant tumor type was lung cancer (73.4%). Although the rate of CAT admissions remained steady during the study period (2009: 5.0% vs 2014: 4.9%, p <0.01), the inpatient mortality was higher among patients admitted with CAT (10.2% vs 9.3%, OR:1.11, CI: 1.08 - 1.13, p < 0.01). The negative effect of VTE on inpatient mortality persisted after adjusting for tumor type, metastatic disease and demographics (OR:1.11, CI: 1.08 - 1.13, p < 0.01). The median length of stay was 5 days in both groups (p<0.01), but the cost was significantly higher among those admitted with CAT (USD33,468 vs USD30,452, p <0.01). Figure 1 summarizes demographics (
A
) and trends (
B
)

Conclusion:
CAT admissions in patients with stomach, pancreas and lung cancer are associated with a higher inpatient mortality and its management is costly. Conceivably, risk stratification based on early mortality may assist in therapy and resources selection to improve outcomes and cost allocation.
Abstract 374: Lack of Fibronectin Containing Extra Domain a Attenuates Acute Myocardial Ischemia/Reperfusion Injury in Hyperlipidemic Mice by Limiting Postischemic Thrombo-inflammation
Authors: Chorawala Mehul , Prakash Prem , Doddapattar Prakash , Dhanesha Nirav , Chauhan Anil
Published: March 2019

Background:
Reperfusion therapy is standard care for patients following acute myocardial infarction. Fibronectin-splicing variant containing extra domain A (Fn-EDA), which is an endogenous ligand for toll-like-receptor 4 (TLR4), is upregulated during vascular hypertension, myocardial injury, and atherosclerosis. An earlier study done in rabbit showed that Fn-EDA accumulates more rapidly (within 24 hrs) in the reperfused hearts. Very little is known about the mechanistic role of Fn-EDA in the pathophysiology of myocardial infarction (MI) in the comorbid condition of hyperlipidemia.

Methods:
Susceptibility to MI outcome was evaluated in hyperlipidemic Apoe
-/-
, Fn-EDA
-/-
Apoe
-/-
, TLR4
-/-
Apoe
-/-
and Fn-EDA
-/-
TLR4
-/-
Apoe
-/-
mice (male and female; 8-10 weeks old) by transient 1-hour ischemia/23 hours of reperfusion (I/R). Myocardial I/R injury outcome was evaluated by measuring infarct area, cardiac Troponin I (cTnI) levels in plasma, postischemic thrombo-inflammation (thrombi and neutrophil influx) and myocyte apoptosis.

Results:
Irrespective of gender, Fn-EDA
-/-
Apoe
-/-
mice (Apoe
-/-
mice expressing Fn deficient in EDA) exhibited smaller infarcts and decreased cTnI levels concomitant with reduced postischemic thrombosis, inflammation and myocyte apoptosis (
P
<0.05 vs. Apoe
-/

-

mice). Genetic ablation of TLR4 attenuated myocardial I/R injury outcome in Apoe
-/-
mice (
P
<0.05 vs. Apoe
-/-
mice), but did not further reduce in Fn-EDA
-/-
Apoe
-/-
mice, suggesting Fn-EDA modulates TLR4-dependent MI exacerbation. Bone marrow transplantation experiments revealed that nonhematopoietic cells-derived Fn-EDA exacerbates myocardial I/R injury through TLR4 expressed on the hematopoietic cell. Infusion of a specific inhibitor of Fn-EDA into Apoe
-/-
mice significantly reduced myocardial I/R injury.

Conclusions:
Hyperlipidemic mice deficient in Fn-EDA exhibit TLR4-dependent reduced myocardial I/R injury that was associated with decreased thrombo-inflammatory response. These findings suggest that targeting Fn-EDA combined with thrombolytic agents could be an effective therapeutic strategy to inhibit myocardial I/R injury in patients with hyperlipidemia.
Abstract 385: Ultracentrifugation and Depletion Methods of High-density Lipoprotein Isolation Yield Particles That Are Functionally Distinct in Some of Their Vasoprotective Functions
Authors: Button Emily , Robert Jerome , Gilmour Megan , Cheema Harleen , Wellington Cheryl
Published: March 2019

The evaluation of the functional capabilities of high-density lipoproteins (HDL) has been shown to be a better predictor of several cardiometabolic diseases than the traditional marker of plasma HDL cholesterol. An important, yet often unconsidered, factor in clinically evaluating HDL function is the method used to purify HDL from plasma. It has been shown that the method of HDL isolation can impact the composition of the HDL produced yet the effect of these compositional changes on HDL function has been explored very little. The evaluation of isolation method is especially important when studying novel HDL functions, such as the vasoprotective functions relevant to Alzheimer’s disease (AD) recently discovered on brain vascular cells and in 3-dimensional brain vessel models. We compared the high-throughput HDL isolation method of polyethylene glycol (PEG) precipitation to the more time-consuming but purer method of sequential density gradient ultracentrifugation (UC) in several of these novel, brain-relevant vasoprotective functions. HDL isolated from young, healthy human plasma by PEG precipitation and UC were equal in their capacity for effluxing cholesterol from macrophages, suppressing TNFα-induced inflammation in brain endothelial cells, and preventing the pathological accumulation of the AD protein amyloid beta (Aβ) in 3D vessel models. However, only HDL isolated by UC, and not PEG precipitation, could induce nitric oxide production and inhibit Aβ-induced inflammation in brain endothelial cells. These findings highlight the importance of HDL isolation method in the composition and function of the HDL produced. Furthermore, that HDL isolation method affected some but not all HDL functions shows that HDL may act on brain vascular cells to protect against AD through more than one pathway.
Abstract 387: Apolipoprotein E Receptor-2 Restricts Cardiomyocyte Proliferation and Controls Heart Size
Authors: Jaeschke Anja , Konaniah Eddy S , Robbins Nathan J , Koch Sheryl E , Rubinstein Jack , Hui David Y
Published: March 2019

Introduction:
Apolipoprotein E receptor 2 (ApoER2) is highly expressed in brain and testes, but also in macrophages, endothelial and vascular smooth muscle cells, as well as heart. Importantly, genome-wide association studies have linked polymorphisms in the LRP8 gene to familial and premature coronary artery disease (CAD) and myocardial infarction (MI). However, the mechanisms by which ApoER2, the protein encoded by the LRP8 gene, affect CAD and MI are incompletely understood.

Hypothesis:
The goal of the current study was to examine the function of ApoER2 in the heart.

Methods and Results:
Morphological analysis revealed an increase in heart size and cross-sectional area with elevated cardiomyocyte proliferation in mice deficient for ApoER2 compared to wild-type control mice. Echocardiography showed a significant increase in left ventricular diameter in ApoER2 KO mice, while no significant differences in fractional shortening and ejection fraction were observed. Loss of ApoER2 attenuated expression of Disabled-2 (Dab2) and Axin2, thereby preventing formation of the destruction complex, resulting in increased levels of β-catenin. Consistent with previous studies that demonstrated that stabilized β-catenin alters expression of the T-box protein Tbx20, ApoER2 deficiency was associated with increased Tbx20 expression, a transcription factor necessary and sufficient to promote cardiomyocyte proliferation.

Conclusions:
Together, these data indicate that ApoER2 promotes β-catenin degradation and modulates Tbx20 expression to restrain cardiomyocyte proliferation and heart size, providing a mechanism by which ApoER2 dysfunction may contribute to premature CAD and MI.
Abstract 398: Gut Microbiota Facilitates Apolipoprotein A1 and HDL Production Through Hepatic TLR5
Authors: Yiu Jensen H , Fung Wai-Lun W , Li Jin , Cheung Jamie , Tso Patrick , Xu Aimin , Woo Connie W
Published: March 2019

Gut microbiota is considered as an external organ as these trillions of commensal microbes in our gut interact with our body to regulate various physiological responses. The pattern and composition of gut bacteria is subject to alteration by our diet. High fat diet not only decreases the β-diversity of gut microbiota but also increases gut permeability, leading to the leak of bacterial products into circulation, which aggravates various diseases including atherosclerosis by escalating inflammation. Paradoxically high fat intake can augment HDL level (J Clin Invest 91:1665-71, 1993). Moreover, gut microbiota was shown to contribute the variation of HDL but not LDL or total cholesterol level in a human study. (Circ Res 117: 817-24, 2015) We speculate that gut microbiota may provide an adaptive protection by upregulating HDL level. In this study, we investigated the possible underlying mechanism of how gut microbiota affects HDL level. C57BL/6J mice were fed a normal chow diet and a high fat diet (HFD, 45% fat kcal) for 10 weeks. Other than decreasing the β-diversity of gut microbiota, HFD increased the portion of flagellated bacteria, resulting in the increase of hepatic but not circulating level of flagellin, the ligand of toll-like receptor (TLR5). Deletion of TLR5 in mice suppressed HFD-stimulated HDL level. Treatment with flagellin in hepatocytes was able to stimulate apolipoprotein A1 (apoA), the primary apolipoprotein on HDL in TLR5-dependent manner. Blocking the downstream signaling of TLR5 by silencing MYD88 blocked the flagellin-induced apoA1 production in wild type hepatocytes. Lipopolysaccharides, another important bacterial product which can activate MYD88 signaling, failed to elicit the same effect. It might be due to the different degree of sensitivity of these TLRs in hepatocytes. In addition, supplementation of flagellin to atherogenic mice (apoE knockout mice) by oral gavage was able to elevate apoA1 and HDL levels in circulation, partially protecting against atherosclerosis. Our data provides a molecular explanation of how commensal bacteria in gut stimulate HDL production upon HFD, and such finding will provide an insight in developing alternative strategy to stimulate HDL production and prevent atherosclerosis.
Abstract 399: Cell, Mice and Human CSF Studies Indicate Impaired Interactions Between ABCA-1 and ApoE4 in ApoE4 Driven Alzheimer’s Disease
Authors: Rawat Varun , Boehm-Cagan Anat , Johansson Jan , Bielicki John , Chui Helena , Harrington Michael , Michaelson Daniel , Yassine Hussein
Published: March 2019

Background:
Loss-of-function mutations in the ATP Binding Cassette A-1 (ABCA-1) transporter and the ApoE4 allele are associated with increased Alzheimer’s disease (AD) risk. We recently reported decreased ABCA-1 activity of cerebrospinal fluid (CSF) from participants with AD. Our aim is to study the interaction of ApoE4 with ABCA-1 activity in astrocytes, in human apoE4 (hApoE4) targeted replacement (TR) mice, and in CSF from humans grouped by APOE genotype.

Methods:
ABCA-1 expression and activity were assessed in astrocytes that express human ApoE isoforms and in hApoE mice. The ability of CSF to activate ABCA-1 was examined in cells. CSF from 59 older individuals with or without cognitive impairment was analyzed for ApoE particle size using native gel electrophoresis, and ABCA-1 cholesterol efflux activity. The effect of the ABCA-1 agonist CS-6253 on ABCA-1 activity and AD pathology was examined.

Results:
ApoE4 vs. apoE3 expressing astrocytes had decreased ABCA-1 expression that was associated with hypolipidated ApoE4. A similar discrepancy for ABCA-1 and apoE lipidation was found between hApoE4 and hApoE3 TR mice. CSF ApoE was resolved in four distinct bands by electrophoresis α
0
(>669 KDa), α
1
(600 KDa), α
2
(440 KDa) and α
3
(232-140 KDa). The amount of total ApoE present in α
0
size was reduced in ε4/ε4 vs ε3/ε3 individuals (3.208 % (SD 0.6156; N=3) vs 8.904 % (SD 0.6156; N=29), p<0.05), whereas total ApoE in α
2
size was increased in ε4/ε4 vs ε3/ε3 individuals (60.68 % (SD 8.207; N=3) vs 37.34 % (SD 16.80; N=31), p<0.05). CSF from ε4/ε4 individuals (N=3) has reduced capacity to induce ABCA-1 mediated cholesterol efflux compared to CSF from non ε4 individuals (N=9) (adjusted efflux 1.288 vs 1.594, p<0.05). CS6253 incubation with CSF increased ABCA1 mediated efflux in hApoE3 and hApoE4 TR mice, respectively. Treatment with the ABCA-1 agonist CS-6253 enhanced CSF cholesterol efflux capacity and reversed cognitive deficits in hApoE4 TR mice.

Conclusions:
ApoE4 was associated with reduced ABCA-1 activity and hypolipidated ApoE4
in vivo
,
in vitro and in CSF
. The ABCA-1 agonist CS6253 improved apoE lipidation in astrocytes, CSF and in hApoE4 TR mice. The findings stress the importance of ABCA-1 in apoE4 driven AD and ABCA-1 activity of CSF as a biomarker in AD.
Abstract 408: ApoE Regulation of Hematopoiesis Suppresses Atherosclerosis by Reducing Adaptive Immunity in Hyperlipidemic Mice
Authors: Bouchareychas Laura , Yamamoto Ryo , Luk Fu Sang , Kim Roy Y , Wong David , Villeneuva Jonathan , Duong Phat , Chung Allen , Nakauchi Hiromitsu , Raffai Robert
Published: March 2019

Background and Purpose:
Apolipoprotein (apo) E suppresses atherosclerosis by exerting a profound control over cells of the innate immune system including monocytes and macrophages. In mice with hyperlipidemia, apoE also prevents an exaggerated proliferation of hematopoietic stem and progenitor cells (HSPC) to further limit the expansion of innate immune cells. However, apoE’s control over hematopoiesis and its subsequent shaping of adaptive immunity remains unexplored.

Methods:
We explored this question by studying HypoE mice deficient in the LDL receptor (
Apoe
h/h
Ldlr
–/–
mice) that display plasma lipid levels similar to those of
Apoe
-/-
Ldlr
-/-
mice when fed a chow diet, but accumulate apoE in plasma and display reduced atherosclerosis. Populations of HSPC were examined in the bone marrow (BM) and spleen, while populations of mature immune cells and their levels of cellular activation were examined in the blood, spleen, lymph nodes and aorta of twenty-week-old mice.

Results:
Our findings show that apoE suppressed the expansion of HSPC in both the spleen and BM of
Apoe
h/h
Ldlr
-/-
mice, which remained similar to levels seen in normal wildtype mice. Specifically, in the spleens of
Apoe
h/h
Ldlr
-/-
mice, apoE reduced the expansion of a population of multi-potent progenitors termed MPP4 responsible for lineages of lymphoid cells. Accordingly,
Apoe
h/h
Ldlr
-/-
mice displayed smaller spleens and lymph nodes that contained fewer myeloid- and lymphoid-derived leukocytes compared to
Apoe
-/-
Ldlr
-/-
mice. The spleens and lymph nodes of
Apoe
h/h
Ldlr
-/-
mice had a higher percentage of T cells that displayed a naïve phenotype, while the percentage of effector T cells was decreased and produced 50% less IFN-γ. Dendritic cells isolated from spleens of
Apoe
h/h
Ldlr
-/-
mice showed reduced levels of CD86 that plays a key role in T cell activation. Digested aortas of
Apoe
h/h
Ldlr
-/-
mice revealed a decrease in both T cell and myeloid cell number that also displayed a lower percentage of the pro-inflammatory CD11b
+
subtype but a higher percentage of the anti-inflammatory CD103
+
subtype.

Conclusion:
Collectively, our findings show that the control of hematopoiesis exerted by apoE results in reduced adaptive immunity to suppress atherosclerosis in hyperlipidemic mice.
Abstract 423: Role of Autophagy in Macrophages During Acute Myocardial Infraction
Authors: Chen Shuang , Shimada Kenichi , Crother Timothy R , Yamashita Michifumi , Arditi Moshe
Published: March 2019

Acute myocardial infarction (AMI) is associated with inflammation and proinflammatory cytokine release, including IL-1β, but the role of NLRP3 inflammasome in cardiac injury remains controversial and requires further investigation. Autophagy, important for stress responses in injury and diseases, is a self-digestion process for the recycling of cytosolic macromolecules and damaged organelles. While autophagy has been previously investigated in cardiac muscle cells in AMI, its role in inflammatory cells is unknown. We generated macrophage (Mφ)-specific Atg16L1 KO mice (
Atg16l1
fl/fl
LysM-Cre) and performed permanent ligation of the left anterior descending artery (LAD) using closed-chest model. 24 hours post ligation, Mφ-specific Atg16L1 KO mice had significantly larger infarction area as measured by myoglobin staining, and more TUNEL staining compared with control groups. This suggested that autophagy in macrophages plays a cardioprotective role during AMI. Since autophagy negatively regulates NLRP3 inflammasome activation and IL-1β secretion, we investigated the role of NLRP3 inflammasome in AMI. NLRP3 KO mice were subjected to LAD ligation and KO mice had less infarction compared with control mice. We also observed reduced infarction in IL-1R1 deficient mice, suggesting that NLRP3 inflammasome and subsequent IL-1β secretion and signaling play a role in AMI pathology. Multiple reports indicate that autophagy associated proteins induction have reduced expression in aged tissues and that autophagy diminishes with aging. We therefore investigated IL-1 signaling blockade with Anakinra (IL-1 receptor antagonist) in aged C57BL/6 mice. Mice were injected with Anakinra daily 10 mg/kg starting 1 h after LAD ligation. While we observed 14% larger infarct size in aged mice (18 months) compared to young mice (12 weeks), Anakinra treatment displayed significantly reduced infarction compared with age matched controls and the reduction of infarction by anakinra treatment was significantly greater in aged mice (75% reduction) compared with young mice (50% reduction). Taken together, these data demonstrate that autophagy plays a protective role in macrophages and NLRP3 and IL-1β mediate pathology, with an increased effect on aged mice during AMI.
Abstract 425: Resistin induced Differential Global Protein Expression in Human Macrophage
Authors: Haider Nezam
Published: March 2019

Background:
Human resistin synthesized by macrophages is shown to be associated with insulin resistance, type 2 diabetes, atherosclerosis, and chronic inflammation. However, there are several missing links related to downstream targets and intracellular signaling pathways involving hyper-resistinemia-associated metabolic disorders. In this study, we used a comparative global protein expression profiling of human macrophage treated with resistin to identify some of these missing links.

Methods:
Human macrophage (Mϕ) derived from THP1 cells were treated with 10ng/ml of human resistin for 48 hrs. Samples of total protein tryptic digest of untreated control and resistin-treated Mϕ were analyzed by HPLC-ESI-MS/MS. Differentially expressed proteins were analyzed by using the Gene Ontology (GO), DAVID, and KEGG databases to characterize their potential functional roles and related pathways.

Results:
Proteins with significant (ρ < 0.05) differences in expression between control and resistin treated groups are shown in Table I. The differentially expressed proteins were mainly involved in metabolic process, cell-cell adhesion, MAPK cascade, protein polyubiquitination, autophagy, and NIK/NF-κB signaling. Majority of the differentially expressed proteins were located in the extracellular exosome, mitochondrion, nucleoplasm, and proteasome complex Table II & III.

Conclusion:
Resistin significantly changed the expression of ∼162 proteins in human macrophage. The differentially expressed proteins are involved in metabolic process, signal transduction pathways, immunological and inflammatory response, exosome and mitochondrial function. This work provides the foundation for future in-depth understanding of resistin-associated metabolic derangement.
Abstract 429: Exercise and Heart Healthy Lifestyle Program also Improve Mental Health
Authors: Ramirez Francisco E , Nedley Neil
Published: March 2019

Background:
Exercise conveys benefits to cardiovascular and mental health. A program that uses exercise is analyzed.

Methods:
Retrospective data from 5221 participants (n=3680, 70% women) that finished the educational program was used, which taught heart healthy lifestyle changes such exercise, plant-based nutrition, stress management among many interventions. The program meet once a week for 8-week, emphasis is given to reach the goal of exercising at least 4 times a week for at least 30 minutes (ideally 60 minutes). Participants completed at baseline, and at the end, the Depression and Anxiety Assessment Test (DAAT), a 75-item questionnaire assessing depression, anxiety, emotional intelligence (EQ), demographic data, and exercise habits.

Results:
At baseline depression was 12 (moderate level) SD 7.59, anxiety of 7 (mild) SD 4.6, EQ 100 SD 15.1 not all the participants were depressed. At baseline 64.8% (n=3385) acknowledged that they didn't do exercise more than 4 times a week on a regular basis. By the end of the 8-weeks, participants mean depression was 6 (moderate) SD 6, anxiety of 3 (none) SD 3.6, EQ 109 SD 14.3 and 46% (n=2402) of them did not exercise more than 4 times a week. There was a significant difference in the depression scores t(5220)=66.44, p<.001 and anxiety scores t(5220)=62.07, p<.001. Table 1 highlights more results. The depression was classified according to DSM-5 into 4 categories as none (0-6), mild (7-10), moderate (11-19) or severe (20 or more).

Conclusion:
Four or more sessions of physical exercise a week was related to improvement in overall health including depression, anxiety and EQ level, even before the program started.
Abstract 433: Difference in pAAV/D377Y-mPCSK9-induced Expression of mPCSK9 Between Male and Female Mice
Authors: Vozenilek Aimee E , Chandran Sunitha , Blackburn Cassidy M , Castore Reneau , Klein Ronald L , Woolard Matthew D
Published: March 2019

Objective:
The recombinant adeno-associated viral vector serotype 8 (AAV8) expressing the gain-of-function mutation of mouse proprotein convertase subtilisin/kexin type 9 (PCSK9) is a new model for the induction of hypercholesterolemia and atherosclerosis development. AAV8 tends to preferentially infect hepatocytes and the liver-specific promoter should ensure expression of PCSK9 results in reduced hepatic low-density lipoprotein receptor levels. However, the tissue distribution of adeno-associated viral vectors can differ between male and female mice. Therefore, we set out to investigate differences in PCSK9 expression and hypercholesterolemia development in male and female mice using the AAV8-PCSK9 model.

Approach and Results:
Male and female C57BL/6 mice were retro-orbitally injected with a low-dose (3x10
10
vector genomes) of AAV8-PCSK9 and fed a high-fat diet for 8 weeks. Inoculation of male mice with low dose AAV8-PCSK9 dramatically elevated both serum PCSK9 and cholesterol levels, which was not observed in female mice. Increasing the inoculation dose of AAV8-PCSK9 threefold (9x10
10
vector genomes) in female mice induced serum cholesterol and PCSK9 concentration to levels equivalent with low-dose inoculated male mice. Although increasing the dose of AAV8-PCSK9 induces a hypercholesterolemia phenotype in female mice, it did not result in an increased amount of AAV8 virus nor increased mRNA expression of mPCSK9 in female livers but did increase mRNA expression of mPCSK9 in the brains of these mice.

Conclusions:
Our data demonstrate that AAV8-PCSK9 inoculation results in differences between male and female mice in the localization and production of mPCSK9. These differences do not hinder the use of female mice in hypercholesterolemia and atherosclerosis studies when AAV8-PCSK9 doses are taken into consideration. However, localization to and production of AAV8-PCSK9 in organs besides the liver in female mice may introduce confounding factors into studies and should be considered during experimental design.
Abstract 442: Loss of
Kiaa1462
, a Coronary Artery Disease Associated Gene, Decreases Atherosclerosis
Authors: Douglas Gillian , Kyriakou Theodosios , Rashbrook Victoria S , Al Haj Zen Ayman , Trelfa Lucy , Mehta Vedanta , Tzima Ellie , Watkins Hugh , Channon Keith
Published: March 2019

Introduction:
KIAA1462
is the only coding gene in a chromosome 10 genomic locus consistently associated with Coronary Artery Disease (CAD) in Genome Wide Association Studies. KIAA1462 has no recognizable functional domains and little homology to other protein families, but has been previously implicated in endothelial cell proliferation and angiogenesis. We used a murine knock out (KO) model to investigate the role of
Kiaa1462
in the development of atherosclerosis.

Methods and Results:
Kiaa1462
global KO mice were generated by replacing exon 3 with a reporter/selection cassette harbouring LacZ.
Kiaa1462
KO mice bred normally with offspring born in the expected Mendelian ratio. X-gal staining of aortas showed
Kiaa1462
expression in vascular smooth muscle cells (VSMC) and endothelial cells. This was confirmed by qRT-PCR with expression of
Kiaa1462
observed in both isolated endothelial cells and VSMC from WT but not KO mice. Loss of
Kiaa1462
did not alter blood pressure or heart rate. Furthermore, no difference in vascular contractile function, or either endothelial cell dependent or independent dilation in the aorta was observed between WT and KO mice. To assess the role of
Kiaa1462
in the development of atherosclerosis we crossed
Kiaa1462
KO mice with hyperlipidaemic ApoE
-/-
mice. Heterozygous breeding pairs were used to generate matched WT, heterozygous and homozygous KO mice. On an ApoE
-/-
background
Kiaa1462
KO mice were no longer born at the expected Mendelian ratio with a significant (~50%) decrease in KO mice alive 21 days after birth. Surviving KO mice were indistinguishable from their littermates with no difference in body or organ weight observed. Atherosclerosis was assessed after high fat feeding for 10 weeks (6 to 16wk of age). Loss of
Kiaa1462
caused a significant decrease in plaque burden in the aortic root of both male and female KO mice vs. WT mice, with no difference observed between WT and heterozygous KO mice. Despite the decrease in plaque size there was no significant difference in macrophage or collagen content between genotypes.

Conclusions:
Our findings support that
Kiaa1462
a GWAS gene candidate plays a critical role in the development of atherosclerosis.
Abstract 471: Influence of Platelet-specific Soluble Guanylyl Cyclase Deficiency on Atherosclerotic Plaque Formation
Authors: Stroth Marlène , Bettaga Noomen , Wobst Jana , Werner Julia , Zimmermann Anna-Sophia , Friebe Andreas , Sager Hendrik B , Schunkert Heribert , Kessler Thorsten
Published: March 2019

Introduction:
The genomic locus harboring the
GUCY1A3
gene has been associated with coronary artery disease (CAD) with the risk variant reducing
GUCY1A3
expression via allele-specific transcription factor binding. The gene encodes the alpha
1
-subunit of the soluble guanylyl cyclase (sGC) which produces the second messenger cyclic guanosine monophosphate (cGMP) upon activation by nitric oxide, thereby influencing the function of vascular smooth muscle cells and platelets.

Aim:
To investigate whether a reduction in sGC expression specifically in platelets influences atherosclerotic plaque formation.

Methods:
By crossbreeding Pf4-Cre and sGC β
1
fl/fl
mice, platelet-specific sGC knockout (PS-sGC
-/-
) mice were generated. These were crossbred with
Ldlr
-/-
mice to generate PS-sGC
-/-
Ldlr
-/-
mice. PS-sGC
-/-
Ldlr
-/-
and
Ldlr
-/-
mice were fed a high-cholesterol diet (HCD) for 10 weeks. Serial sections of the aortic root were analyzed by Masson-Trichrome staining. Inflammatory cells in aortae were assessed using flow cytometry.
In vitro
adhesion assays using wildtype endothelial cells (EC) and monocytes were performed in presence of plasma of activated platelets.

Results:
PS-sGC
-/-
Ldlr
-/-
(n=13) mice displayed enhanced total plaque size compared to
Ldlr
-/-
(n=15) mice (247·10
3
vs. 190·10
3
μm
2
, p<0.05). After HCD, more neutrophils (747 vs. 448, p<0.05) and Ly6C
high
monocytes (366 vs. 207, p<0.05) were detected in PS-sGC
-/-
Ldlr
-/-
compared to
Ldlr
-/-
mice indicating enhanced recruitment of these cells in PS-sGC
-/-
Ldlr
-/-
mice. In
in vitro
adhesion assays, incubation of mouse aortic EC and monocytes with plasma from PS-sGC
-/-
platelets resulted in enhanced adhesion of monocytes to EC compared to plasma from wildtype platelets (13%, p<0.01).

Conclusion and Outlook:
Platelet-specific knockout of sGC led to increased atherosclerotic plaque formation and inflammation.
In vitro
adhesion assay results point to an enhanced stimulation of leukocyte adhesion to endothelial cells in PS-sGC
-/-
mice. Hence, factors released by platelets secondary to reduced sGC expression could mediate increased risk of CAD. Inhibiting platelet activation in carriers of the human risk variant might therefore be a promising therapeutic strategy to reduce risk of CAD.