bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 548: Separation of Lipoprotein Particles Utilizing Asymmetric Flow Field-flow Fractionation and Quantitation of Apolipoprotein L1 in Human Plasma with IMER-UPLC-MS/MS
Authors: Andrews Michael , Kuklenyik Zsuzsanna , Williamson Yulanda , Bierbaum Kevin , Schieltz David , Parks Bryan , Toth Christopher , Gardner Michael , McWilliams Lisa , Lehtikoski Anthony , Rees Jon , Barr John
Published: March 2019

Apolipoprotein L1 (ApoL1) is an intriguing protein and was shown to be involved in numerous diseases states such as chronic kidney disease (CKD), cardiovascular diseases(CVD), Human Immunodeficiency Virus associated Nephropathy (HIVAN), systemic lupus erythematosus (SLE) (associated collapsing glomerulopathy), and type 2 diabetes(especially for people of African descent). People of African ancestry were shown to have either one or two alleles which contribute to their susceptibility of diseases related to CKD. ApoL1 is characterized as a HDL binding protein. In this work we studied ApoL1 binding to HDL by using a gentle size fractionation technique, asymmetric flow field-flow fractionation (AF4), and liquid chromatography (LC) with quantitative tandem mass spectrometry (MS/MS) detection. We injected 50 uL of whole plasma onto the AF4 system, where smaller HDL particles eluted first followed by the larger HDL, and then by LDL, IDL and VLDL particles. The lipoproteins were separated into 40 fractions and quantitatively analyzed using on-line trypsin digestion coupled with LC-MS/MS analysis. We analyzed plasma samples both with and without purification by UC. When HDL particles were separated by UC, apoL1 was found in the higher density HDL fractions, as was also found by previous studies. When HDL separated using AF4 with or without UC, apoL1 eluted in the larger HDL size region (12-13 nm). With UC vs. without UC, the apoL1 peptide signal intensities were reduced by more than 50%, indicating significant loss of apoL1 from the surface of HDL particles during UC. Our data on apoL1 provides further evidence for the fact that size and density are not directly interconvertible physical characteristics of lipoproteins. Furthermore, our data shows that apoL1 is also an example of exchangeable apolipoproteins whose binding can be significantly diminished due to the effect of intense shear forces during UC separation. This work also demonstrates the advantages of utilizing AF4-IMER-UPLC-MS/MS methodology to separate lipoprotein particles and study of their apolipoprotein composition.
Abstract 560: Protein and Lipid Composition of HDL Particles
Authors: Niisuke Katrin , Kuklenyik Zsuzsanna , Vaisar Tomas , Asztalos Bela F
Published: March 2019

Objective:
HDL cell-cholesterol efflux capacity is influenced by both the concentration and the functionality (efflux capacity per particle) of HDL particles. It is assumed that the lipid and protein composition of HDL particles significantly influence their functionality; however there is little data to support that. Our aim was to determine the protein and lipid composition of individual HDL particles to better understand the large variability observed in HDL functionality studies.

Approach:
ApoA-I-containing HDL particles (preβ-1, α-4, α-3, α-2, and α-1) were separated by non-denaturing 2d-PAGE, electro-eluted from the gel, and purified by immuno-affinity chromatography using anti-human apoA-I IgG. The protein and lipid composition of the particles were assessed by mass-spectrometry. The number of apoA-I molecules in each particle was determined by cross-linking experiments.

Results:
The number of protein species, associated with HDL particles, increased with particle size: the largest (α-1) particles carried more than 50, while the smallest (preβ-1) particles carried only a few different proteins. ApoE was present in trace amount, if any, in these HDL particles. The apoA-II/apoA-I ratio was very low in the α-1, α-4, and preβ-1 particles but substantially higher in the α-2 and α-3 particles. The maximum number of apoA-I molecules was 4 in α-1 and 3 in α-2, α-3, and α-4 particles. The largest (α-1) particles contained significantly more lipid molecules, especially triglycerides, for each apoA-I molecule compared to smaller particles. The lipid composition of the intermediate-size α-2 and α-3 particles was similar. In contrast to larger HDL particles, the small α-4 particles were enriched in sphingomyelin. Interestingly, the very-small preβ-1 particles contained about equal amounts of polar (phospholipids and free cholesterol) and non-polar (cholesteryl ester and triglycerides) lipids. Phospholipids were represented only by sphingomyelin. These findings contradict previous models that indicated that each preβ-1 particle contained one or two apoA-I and a few polar lipid molecules.

Conclusions:
Both the protein and the lipid composition of HDL particles are size dependent. Further studies are needed to determine how the protein and lipid composition of HDL particles influence HDL functions.
Abstract 583: Targeting of Macrophage Netrin-1 Expression Promotes Plaque Regression and Resolution of Chronic Inflammation in Atherosclerosis
Authors: Schlegel Martin , Sharma Monika , Afonso Milessa , Beckett Lauren , Babunovic Susan , Moore Kathryn J
Published: March 2019

Chronic inflammation in atherosclerosis is driven by the accumulation of cholesterol-laden macrophages in the arterial wall. These pro-inflammatory macrophages persist in this site, and sustain local and systemic inflammation. Recently, we reported that the neuronal guidance protein netrin-1 plays a pivotal role in the retention of macrophages in plaques and the progression of atherosclerosis. To test whether targeting netrin-1 in established atherosclerotic plaques could promote the resolution of chronic inflammation in atherosclerosis and/or induce plaque regression, we developed mice in which macrophage netrin-1 expression could be inducibly deleted by tamoxifen administration (Ntn1
fl/fl
CX3CR1
CreER2+
mice). We first induced atherosclerosis in Ntn1
fl/fl
CX3CR1
CreER2+
and control Ntn1
fl/fl
CX3CR1
CreER2-
using a recombinant AAV-vector overexpressing PSCK9 and Western diet feeding. After 20 weeks, 10 mice from each group were sacrificed for baseline plaque measurements, while the remaining mice were switched to chow diet to stop further progression of atherosclerosis and treated with tamoxifen (n=14/group). After 4 weeks, mice were sacrificed to assess the effects of macrophage-specific netrin-1 deletion on plaque size and composition. Analysis of peritoneal macrophages confirmed robust deletion of netrin-1 in tamoxifen treated Ntn1
fl/fl
CX3CR1
CreER2+
but not control Ntn1
fl/fl
CX3CR1
CreER2-
mice. Macrophage-specific deletion of netrin-1 caused a 30% reduction in plaque burden in the aortic arch as measured by en face analysis, compared to control mice. While we observed no change in aortic macrophage content, macrophage-specific netrin-1 deletion was associated with a shift in effector T cell populations in the aorta: we observed a decrease in Th1 cells and an enrichment of Th2 and Treg cells in aortic plaques of Ntn1
fl/fl
CX3CR1
CreER2+
compared to control Ntn1
fl/fl
CX3CR1
CreER2-
. Furthermore, deletion of macrophage netrin-1 expression was associated with a reduction of systemic IL-1β levels. Collectively, these data suggest that targeting macrophage expression of netrin-1 in established atherosclerosis fosters a local pro-resolving and atheroprotective T cell phenotype in plaques and reduces systemic inflammation.
Abstract 585: Card9 Deficiency Accelerates Experimental Atherosclerosis
Authors: Zhang Yujiao , Zeboudj Lynda , Joffre Jeremie , Vandestienne Marie , Taleb Soraya , Laurans Ludivine , Tedgui Alain , Mallat Ziad , Sokol Harry , Ait-Oufella Hafid
Published: March 2019

Introduction:
There are accumulating evidences that innate and adaptive immunity play a major role in the development of atherosclerosis. Pattern-recognition receptors (PRRs) engagement including Toll-like receptors and Dectins are involved in the modulation of immune responses and atherosclerosis development but little is known about downstream signaling pathways. Card9 for Caspase recruitment domain-containing protein-9, is an adaptor protein expressed by antigen presenting cells required for PRRs-induced activation of myeloid cells. We hypothesized that Card9 pathway regulates systemic immune response and impacts on the development of atherosclerosis.

Method and results:
To evaluate the effect of Card9 deficiency on experimental atherosclerosis, Ldlr
-/-
mice were lethally irradiated and reconstituted with Card9
-/-
or Card9
+/+
bone marrow cells and put under a high fat diet during 8 weeks. Animal weight and cholesterolemia were not different between groups. We observed an increase of atherosclerosis plaque size in the aortic sinus in chimeric Ldlr
-/-
Card9
-/-
mice compared to chimeric Ldlr
-/-
Card9
+/+
mice (+32%, P=0.04). A more inflammatory plaque phenotype was found in chimeric Ldlr
-/-
Card9
-/-
mice compared to control mice with an increase in both macrophage accumulation (+86%, P=0.0005) and necrotic core size (+102%, P=0.006). Card9 deficiency induced a deviation of the systemic immune response toward a pro-inflammatory profile. Lps/Ifn-γ-stimulated Card9
-/-
bone marrow-derived macrophages (BMDM) produced less IL-10 (-22%, P<0.05) than Card9
+/+
BMDM. Lps/Ifn-γ-stimulated splenocytes from chimeric LDLr
-/-
Card9
-/-
mice produced more IL-12p70 (+151%, P<0.01) than splenocytes from control mice. Anti-CD3 stimulated CD4
+
T cells from chimeric Ldlr
-/-
Card9
-/-
mice produced less Ifn-γ (-92%, P<0.05) and IL-17A (-100%, P<0.05) than control CD4
+
T cells. A second atherosclerosis mouse model ApoE
-/-
Card9
-/-
confirmed the protective role of Card9 with an increase in both atherosclerosis plaque size and macrophage accumulation in ApoE
-/-
Card9
-/-
mice compared to ApoE
-/-
Card9
+/+
mice.

Conclusion:
Card9 deficiency accelerated atherosclerosis development in mice and induced a more inflammatory plaque phenotype.
Abstract 598: Monounsaturated Fat Reduces Foamy Monocyte Formation and Atherosclerosis Development in Ldlr
-/-
Mice Compared to Western High Saturated Fat Diet
Authors: Lian Zeqin , Perrard Xiao-yuan D , Peng Xueying , Joe L. Raya , Hernandez Alfredo A , Johnson Collin G , Lagor William , Hoogeveen Ron , Simon Scott I , Sacks Frank , Ballantyne Christie , Wu Huaizhu
Published: March 2019

Monounsaturated fat (MUF)-rich Mediterranean-type diet (MedD) has been reported to improve atherosclerotic outcome in clinical studies, but the underlying mechanism is ill defined. Circulating foamy monocytes (FMs, monocytes with intracellular lipid droplets), which are CD11c
+
and highly adherent to inflamed endothelium, contribute to atherosclerosis development. In the present study, we investigated the influence of MedD on FM formation and its contribution to atherosclerosis in mice. LDLR
-/-
mice were fed MedD with high cholesterol (MedD [w/w, 21% total fat from olive oil and nut] containing 2.6% saturated fat and 13.4% MUF; 0.2% cholesterol) or western diet (WD, 21% milkfat-containing 13.3% saturated fat and 5.9% MUF; 0.2% cholesterol), with normal diet (ND) as control. Lesion area of the whole aorta examined by oil red staining at 3 months was significantly reduced in mice on MedD, compared to WD. Although plasma triglyceride levels were lower in mice on MedD than on WD, the free fatty acid profile indicated that MUFs concentration in plasma significantly increased in mice on MedD. Further, FMs from mice on MedD circulated at lower proportions and exhibited lower side scatter (SSC) than WD, indicating less lipid accumulation. Lipid accumulation in FMs from mice on WD accelerated their conversion from CD11c
-
/CD36
+
to CD11c
+
/CD36
+
compared to mice on ND. In contrast, this accelerated conversion did not occur in mice on MedD. Compared to WD, MedD reduced the number of firmly arrested CD11c
+
monocytes on vascular cell adhesion molecule-1 and E-selectin coated coverslips detected in an ex-vivo shear flow assay. Similarly, fewer CD11c
+
macrophages were observed in the lesion of aortic sinus in mice on MedD than on WD. In summary, compared to WD high in saturated fat and cholesterol, MedD high in MUF and cholesterol lowered triglyceride levels, inhibited foamy monocyte formation and adhesion, and reduced atherosclerosis in LDLR
-/-
mice.
Abstract 618: Identification of Novel Disease Genes for Metabolic Syndrome
Authors: Mani Arya , Bhat Neha , Esteghamat Sahar
Published: March 2019

Factors that underlie the association of diverse traits of the metabolic syndrome have remained largely unknown. The strong heritable nature of this condition provides an exceptional avenue for discovery of the disease mechanisms by using modern techniques of human molecular genetics. Strikingly, efforts by genome-wide association studies to identify common variants that show association with two or more traits have largely failed. Growing evidence indicates excess of rare genetic variants through recent exponential population growth, raising the possibility for their causal role in common diseases. With the advent of high throughput sequencing the power for identification of functional rare variants has dramatically increased. By biasing for extreme phenotypes and utilizing traditional method of segregation analysis we have discovered a number of disease genes for metabolic syndrome and coronary artery disease. A major benefit of this strategy is that true causal relationships has been established between rare variations in a gene, its cognate pathways and the disease of interest. These, in turn, have provided fundamental new insight into pathogenesis of poorly understood diseases such as plaque erosion, and logical starting points for identifying new targets and pathways for therapeutic intervention. By dissecting cognate pathways, we have identified novel targets, including Dyrk1B kinase, a novel incretin and components of Wnt signaling pathway to treat CAD, diabetes and hyperlipidemia and have implemented those succesfuly in animal models.
Abstract 619: Regulatory Mechanism of Lmod1 Mediated Predisposition to Coronary Artery Disease
Authors: Nanda Vivek , Wang Ting , Pjanic Milos , Liu Boxiang , Quertermous Thomas , Miano Joseph , Leeper Nicholas , Miller Clint L
Published: March 2019

Atherosclerotic coronary artery disease (CAD) continues to be the leading cause of mortality and morbidity worldwide, with an estimated 40% of one’s lifetime risk attributed to genetic factors. Meta-analysis of genome-wide association studies implemented to identify this risk in human populations, has now identified rs2820315 (P=7.7E-10; OR=1.05), in the smooth muscle cell-restricted gene, Leiomodin 1 (LMOD1), as the leading genetic polymorphism associated with CAD. However, the causal mechanism by which this polymorphism is responsible for predisposition to CAD remains to be identified. Expression quantitative trait loci (eQTL) mapping in GTEx and STARNET databases revealed that carriers of the risk allele, rs34091558, which is in tight linkage disequilibrium with rs2820315, display significantly attenuated LMOD1 expression in vascular tissues than carriers of the ancestral allele. Allelic expression imbalance analyses in heterozygous HCASMC donors further demonstrated cis-acting effects of the polymorphism on LMOD1 gene expression. To determine the mechanism responsible for this reduction in LMOD1 expression, we performed position weight matrix (PWM) motif analyses and found that rs34091558 disrupts the binding site of a transcription factor called forkhead box O3 (FOXO3). Subsequent chromatin immunoprecipitation and reporter assays demonstrated reduced FOXO3 binding and transcriptional activity by the risk allele in cultured HCASMCs. Platelet-derived growth factor BB (PDGF-BB) stimulation also significantly reduced LMOD1 expression coincident with FOXO3 knockdown. Finally, both gain and loss-of-function for FOXO3 and LMOD1 in HCASMC delineated a regulatory circuit by which LMOD1 regulates SMC proliferation, migration and contraction, characteristic features of atherosclerotic lesion progression. Taken together, these results provide compelling functional evidence that: 1) rs3091558 is associated with reduced LMOD1 expression, 2) this reduction appears to be mediated through the inhibition of FOXO3 binding and 3) changes in vessel wall processes through LMOD1 dysregulation may partially explain the heritable risk for CAD.
Abstract 620: miRNA Nanoparticles Targeting Cholesterol Efflux: a Promising Tool for the Treatment of Atherosclerosis
Authors: Nguyen My-Anh , Susser Leah , Gadde Suresh , Rayner Katey
Published: March 2019

The prevention and treatment of cardiovascular diseases (CVD) has largely focused on lowering circulating LDL cholesterol, yet a significant burden of atherosclerotic disease remains even with low LDL. Recently, microRNAs (miRNAs) have emerged as exciting therapeutic targets for CVD. miRNAs are small noncoding RNAs that post-transcriptionally regulate gene expression by degradation or translational inhibition of target mRNAs. A number of miRNAs have been found to modulate all stages of atherosclerosis, particularly those that promote cholesterol efflux from lipid laden macrophages in the vessel wall. However, one of the major challenges of miRNA-based therapy is to achieve tissue-specific, efficient and safe delivery of miRNAs
in vivo
.

Objective:
We therefore sought to develop chitosan/miRNA nanoparticles, deliver them to the plaque, and determine if these miRNAs can promote cholesterol efflux to decrease atherosclerosis.

Results:
We conjugated negatively charged miRNAs with tripolyphosphate (TPP) to support crosslinks between polymeric and nucleic acid units, which were then mixed with varying ratios of chitosan polymer to form nanoparticles that ranged from 150-180nm in size. We next optimized the efficiency of intracellular delivery of different chitosan/miRNA ratios to mouse macrophages (MΦ). We find chitosan nanoparticles can protect as well as transfer exogenous miR-33 to naïve MΦ and reduce mRNA and protein expression of its target gene, ABCA1, confirming that miRNAs delivered via nanoparticle can escape the endosomal system and function in the RISC complex. Because ABCA1 plays a key role in stimulating the efflux of cholesterol from MΦ, we also confirmed that MΦ treated with chitosan/miR-33 nanoparticles exhibited reduced cholesterol efflux to Apolipoprotein A1, further confirming functional delivery of the miRNA. Using this formulation, we have developed a panel of miRNA nanoparticles delivering miRNAs that enhance ABCA1 expression and promote cholesterol efflux.

Conclusions:
miRNAs can be efficiently delivered to macrophages via nanoparticles where they can function to regulate ABCA1 expression and cholesterol efflux, suggesting that these miRNA-nanoparticles can be used
in vivo
to target atherosclerotic lesions.
Abstract 622: Expression of Fatty Acid Binding Protein-4 in Atherosclerotic Watanabe Heritable Hyperlipidemic (WHHL) Rabbit Aortas
Authors: Prathipati Priyanka , Walton Brian
Published: March 2019

Background:
Atherosclerotic diseases are among the prime cause of mortality globally. Proper animal model that can resemble the lipid metabolism and atherosclerotic lesions in humans is key to study the pathophysiology of the disease. One of the most famous of these atherosclerotic animal models is Watanabe heritable hyperlipidemic (WHHL) rabbits. They are deficient of LDL receptors, hence develop spontaneous hypocholesterolemia and atherosclerosis. Previous studies have demonstrated the importance of Fatty acid binding protein-4 (FABP4), a lipid chaperon that plays an important role in atherosclerotic diseases. However, FABP4 expression or role in WHHL rabbits was not investigated yet. Hence, we hypothesis that WHHL rabbits express FABP4 in their aortas and targeting FABP4 acts as a novel target to treat atherosclerosis.

Materials and Methods:
Two WHHL rabbits of greater than one year old were sacrificed and aortas were isolated. One of the aorta was flash frozen and other was formalin fixed. Proteins were extracted from frozen section. Western blot was performed to estimate the concentration of FABP4. Immunohistochemistry was performed on the formalin fixed tissue to analyze the expression of FABP4.

Results:
WHHL aortas having noteworthy amount of atherosclerotic plaques expressed FABP4 supporting the importance of FABP4 in cardiovascular diseases.

Conclusion:
WHHL rabbits which are model of atherosclerosis expresses FABP4 in their aortas thus paving the way to use this model to target FABP4 for treating atherosclerosis.
Abstract 631: Reduced Liver X Receptor Alpha S198 Phosphorylation in Bone Marrow Protects against Atherosclerosis and Obesity in Mouse Models
Authors: Voisin Maud , Shrestha Elina , Barrett Tessa J , Rollet Claire , Chang Hye Rim , Ruoff Rachel , Goldberg Ira J , Fisher Edward A , Garabedian Michael J
Published: March 2019

Accumulation of cholesterol-loaded macrophages in arterial walls leads to atherosclerosis, the most common cause of cardiovascular disease. Obesity is a major risk factor for atherosclerosis, and both share pathophysiological features. Indeed, high lipid concentrations and inflammation play key roles in the etiology of both diseases, and this is mediated by alterations in macrophage signaling and gene expression. For example, increased intracellular cholesterol in macrophages activates the Liver X Receptor alpha (LXRα), which plays a central role in reverse cholesterol transport by inducing genes involved in cholesterol efflux and transport. LXRα also responds to environmental signals, such as cholesterol overload, via phosphorylation at serine 198 (pS198). LXRα pS198 selectively regulates the expression of genes that promote inflammation, cell movement, and lipoprotein metabolism, which fosters both atherosclerosis and obesity.

However, whether LXRα phosphorylation impacts progression of atherosclerosis or affects adipose tissue has not been determined. To examine a role of LXRα phosphorylation in atherosclerosis and obesity, we developed a LXRα S198 phosphorylation-deficient knock in mouse (S198A). LDLR-/- mice were reconstituted with bone marrow from LXRα WT or S198A mice and placed on a western diet for 16 weeks. Aortic plaques and perigonadal adipose tissue were histologically analyzed and plasma characteristics were measured. Compared to WT, S198A increased anti-atherogenic HDL (69.2 vs 98.8 mg/dL; p=0.0019) and lowered glucose levels (173.2 vs 149.1 mg/dL; p=0.0019), showed a trend toward decreased plaque macrophage content (32.1 vs 28.7 % plaque area; p=0.085), and reduced macrophage recruitment (# entering cells: 16.6 vs 11.1; p=0.0048), plaque macrophage proliferation (36.6 vs 20.1; p<0.0001), and apoptosis (25.4 vs 14.4; p=0.0002). By FACS, the number of inflammatory adipose tissue macrophages (Cd11c+) was reduced in S198A (28.5 vs 21.5% of total F4/80+ cells; p=0.05). We also observed that the adipocytes were smaller in S198A (3570 vs 2471 μm
2
; p=0.01), consistent with a lean phenotype. This suggests that reducing LXRα pS198 in bone marrow promotes a healthier metabolic environment for both atherosclerosis and obesity.
Abstract 639: Clinical, Genetic, and Epigenetic Characteristics of Prolonged Postprandial Lipemia
Authors: Al-Tobasei Rafet , Irvin Marguerite R , Tiwari Hemant K , Hopkins Paul N , Ordovas Jose M , Absher Devin M , Arnett Donna K , Aslibekyan Stella
Published: March 2019

Postprandial lipemia (PPL), defined as the series of metabolic events occuring after ingestion of fat, plays a key role in atherogenesis. While the plasma lipid response to meals is extremely variable, the determinants of PPL variation remain poorly understood. Using data from the Genetics of Lipid Lowering Drugs and Diet Network (GOLDN, n=1039), we investigated clinical, genetic, and DNA methylation correlates of prolonged PPL. Participants were administered a standardized high-fat milkshake and blood samples were taken at baseline, 3.5, and 6 hours post-meal. A prolonged PPL response was defined as a binary trait: 1 if plasma triglycerides at 6 hours exceeded the level at 3.5 hours, 0 otherwise. Genome-wide sequence and methylation variation was quantified using next generation sequencing and Illumina 450K methylation array in CD4+ T-cells, respectively. Approximately one third (n=312) of GOLDN participants exhibited a prolonged PPL response to the high-fat challenge. Prolonged PPL was significantly associated with increasing age (P= 1.0x10
-6
), waist circumference (P= 0.02), fasting glucose (P=0.006), low-density lipoprotein cholesterol (P= 0.04), and fasting triglycerides (P= 1.9x10
-8
), and inversely associated with high-density lipoprotein cholesterol (P=0.0003). Participants with prolonged PPL were significantly more likely to have prevalent coronary heart disease (P=0.0002) and diabetes mellitus (P= 1.5x10
-5
). No genetic variants were significantly associated with PPL at the genome-wide level, in either single variant or gene-based tests. However, a DNA methylation variant in the
COG5
gene, encoding a component of the oligomeric Golgi complex that has been previously shown to be required for intestinal lipid absorption in a zebrafish model, emerged as the top epigenetic determinant of prolonged PPL response (P= 2.5x10
-7
) in a mixed effects model adjusting for age (linear and quadratic), sex, study site, smoking, alcohol intake, T-cell purity, and familial relationships. Upon successful replication in independent cohorts and/or functional validation, the
COG5
finding can be considered a novel epigenetic target in dyslipidemia, with potential to inform future research efforts and personalized treatment approaches.
Abstract 643: Atherosclerosis Susceptibility in the Collaborative Cross
Authors: Bennett Brian J , Rindy Alexa , Albright Jody , Yam Phoebe , VerHague Melissa
Published: March 2019

Objective:
The Collaborative Cross (CC) is a large recombinant inbred mouse population, generated from elaborate intercrosses of C57BL6/J, A/J, NOD/ShiLtJ, NZO/HiLtJ, WSB/EiJ, CAST/EiJ, PWK/PhJ, and 129S1/SvImJ mouse strains, useful for complex trait mapping and systems genetics. The CC population has tremendous genetic diversity, containing approximately 45 million segregating single-nucleotide polymorphisms (SNPs). The purpose of this study was to characterize atherosclerosis susceptibility in an initial cohort of Collaborative Cross strains.

Methods and Results:
We obtained 5 females from 20 CC lines available at the University of North Carolina and placed them on a low-fat, synthetic diet (AIN-76) for 2 weeks. Following two weeks of diet, all mice were assessed for plasma cardio-metabolic risk factors. The mice were then randomly assigned to cages and placed on a synthetic high-fat, cholic acid (HFCA) diet that contained 20% fat, 1.25% cholesterol, and 0.5% cholic acid. Aortic root atherosclerosis and cardio-metabolic risk factors were quantitated following 16 weeks of HFCA diet feeding. Atherosclerotic lesion size ranged from completely resistant (strains C004/TauUnc and CC037/TauUnc) to moderate lesion development in a majority of several strains. Notably all mice from strain CC063/Unc died prior to 20 weeks of age, while strain CC028/GeniUnc was highly susceptible to lesion development with an average lesion size of 105,781 ± 17,370 um
2
. As expected, strain is a significant contributor to atherosclerosis susceptibility (one way Anova analysis, p<8.23x10
-08
). Similar effects were observed for circulating cholesterol with strains C004/TauUnc and CC037/TauUnc having relatively low circulating total cholesterol after HFCA feeding (187 and 198 mg/dl respectively) to > 500 mg/dl in strains CC006/UNC, CC011/Unc and CC036/Unc. Circulating cholesterol and atherosclerosis were significantly correlated (Spearman rho>0.30, p< 0.007).

Conclusion:
These results suggest that selective crosses between various CC strains should yield loci associated with atherosclerosis. High-resolution genome-wide association analysis for atherosclerosis may be feasible when more Collaborative Cross strains become available.
Abstract 648: Resident S100β
+
/Sca1
+
Multipotent Vascular Stem Cells Undergo Myogenic and Vasculogenic Differentiation
In Vitro
Authors: Burtenshaw Denise , Fitzpatrick Emma , Liu Weimin , Morrow David , Redmond Eileen M , Cahill Paul A
Published: March 2019

Arteriosclerosis is an important age-dependent disease that encompasses atherosclerosis, in-stent restenosis, autologous bypass grafting and transplant arteriosclerosis. Vascular smooth muscle (vSMC)-like accumulation is a key event leading to intimal-medial thickening (IMT), vessel remodelling and an important marker of subclinical disease. Vascular stem cell progeny in addition to de-differentiated SMC and SMC derived from endothelial-mesenchymal transition (EndoMT) are all reported to contribute to IMT as they become activated/dysfunctional, differentiate down vascular and myeloid lineages and subsequently dictate, in-part, vessel remodelling. In this study we examined the multipotent potential of a specialised population of rat adult resident multipotent vascular stem cells (rMVSC) located within the vessel wall and their capability to differentiate down both myogenic (muscle) and vasculogenic (endothelial) lineages when given the appropriate stimulus. Using Sca1-eGFP transgenic mice,
in vivo
, there was a significant increase in the number of Sca1
+
cells within the intima of the left carotid artery (LCA) following partial carotid artery ligation-induced injury after 3 days that co-localised with endothelial nitic oxide synthase (eNOS) and CD31
+
positive cells, when compared to the sham-operated control vessels and the contralateral right carotid artery (RCA). The number of Sca1-eGFP
+
cells significantly increased over time within the adventitial, medial and neointimal layers following ligation-induced injury after 7 and 14 days, respectively. In vitro, S100β/Sca1
+
rat MVSCs cultured in vasculogenic inductive media for 7 days underwent differentiation to an endothelial cell phenotype characterised by the appearance of a cobblestone morphology and increased eNOS expression. In contrast, MVSC exposed to media supplemented with TGF-β1 for 7 days underwent myogenic differentiation to SMC. These data suggest that resident S100β/Sca1
+
MVSCs are capable of both myogenic and vasculogenic differentiation depending on the inductive stimulus and may contribute in part to intimal medial thickening (IMT) and endothelial regeneration following injury
in vivo.
Abstract 652: MicroRNA-29b Mediates the Chronic Inflammatory Response in Radiotherapy-induced Vascular Disease
Authors: Winski Greg , Eken Suzanne M , Christersdottir Tinna , Sangsuwan Traimate , Jin Hong , Chernogubova Ekaterina , Pirault John , Sun Changyan , Simon Nancy , Winter Hanna , Tornvall Per , Haghdoost Siamak , Hansson Göran K , Halle Martin , Maegdefessel Lars
Published: March 2019

Radiotherapy is an established therapeutic method in many different cancer types. Its success raises a new problem, namely radiotherapy-induced vascular disease (vRTx), which typically manifests as coronary disease, heart failure or carotid stenosis. A chronic inflammatory response likely underlies vRTx, involving complex processes, such as vascular smooth muscle cell de-differentiation, oxidative stress, and ECM remodeling. These processes are tightly regulated and thus far difficult to influence therapeutically. In other diseases, a set of microRNAs (miRs) is known to orchestrate these processes. We hypothesized that they similarly could play a role in vRTx and be targets for treatment or prevention of the disease.

We performed qRT-PCR screening of 11 pre-selected miRs, which identified miR-29b as significantly decreased in irradiated arteries collected from patients undergoing free tissue transfer reconstruction, compared with non-irradiated arteries from the same patient (n=15). In a vascular biology context, miR-29b is known as inhibitor of collagen- and ECM associated mRNA targets, and has been shown to play a detrimental role in aneurysm disease and advanced atherosclerotic lesions.

Consistent with human tissue data, vascular smooth muscle and endothelial cells
in vitro
receiving two radiation doses of 2 Gy, showed decreased miR-29b upon 24 hours after exposure. In these irradiated SMCs, miR-29b induction reduced soluble collagen levels, while inhibition further increased them.

Array-based tissue gene expression analysis showed that Pentraxin-3 (PTX) and dipeptidyl-peptidase 4 (DPP4), both targets of miR-29b and pivotal in inflammation and adverse wound healing, were downregulated in the same patient cohort. Carotid arteries of
Apoe
-/-
mice treated with miR-29b mimic, 24h before and 24h after irradiation (14 Gy to the upper chest and neck), displayed a downward trend (non-significant) in
Ptx3
and
Dpp4
gene expression.

Our results suggest that miR-29b overexpression therapy could have a place in the prevention of vRTx. To further strengthen this conclusion, additional
Apoe
-/-
mice irradiation experiments are currently ongoing, to further establish PTX3 and DPP4 as mediators of anti-inflammatory and anti-fibrotic strategies.
Abstract 653: Placental Growth Factor mediates Splenic Sympathetic Overdrive induced by Obesity and induces hypertension
Authors: Carnevale Daniela , Lori Andrea , Perrotta Marialuisa , Carnevale Lorenzo , Pallante Fabio , Iodice Daniele , Carnevale Raimondo , Cifelli Giuseppe , Lembo Giuseppe
Published: March 2019

Obesity is an epidemic condition associated with several cardiovascular comorbidities, as hypertension. Although the molecular mechanisms related to obesity-induced hypertension are not well understood, the sympathetic nervous system (SNS) overactivation typically accompanying obesity has been ascribed as one of the main culprits. Thus far, mechanistic studies focused on the effects of sympathetic overdrive on peripheral resistances. Recent data showing that SNS activation also dictates immune responses involved in hypertension, made us hypothesize whether obesity induced by high fat diet (HFD) could recruit splenic SNS efferent. Thus we subjected WT mice to HFD, as compared to low fat diet (LFD), and monitored the hypertensive response. Blood pressure (BP) started to rise after two months, and hypertensive disease became clearly manifest within 4 months. To gain mechanistic insights responsible for BP increase, we assessed peripheral SNS activation at a time point preceding overt hypertension (i.e. 2 months after HFD). We found elevated circulating noradrenalin levels in HFD mice and, more important, a specific pattern of activated splenic sympathetic overdrive, as recorded by microneurography. WT mice subjected by surgical removal of the left celiac ganglion (CGX) where the splenic nerve originate, before receiving HFD, were protected from hypertension, despite developing obesity the same. The fact that splenectomized mice showed an overlapping response to HFD, supported the conclusion that the sympathetic-immune axis is crucial for HFD-induced hypertension but not for weight regulation. To look for molecular determinants, we analyzed the expression of Placental Growth Factor (PlGF), previously identified as a neuroimmune mediator of other hypertensive challenges. PlGF was significantly overexpressed in the spleen upon HFD, but only when SNS innervation was intact, as CGX mice failed to induce its expression. In the end, PlGF KO mice subjected to HFD, although becoming obese as much as WT did, were protected from hypertension. Overall our results demonstrate that a splenic neuroimmune drive is crucial to allow the onset of hypertensive disease associated with obesity, although having no effect in body weight increase in response to HFD.
Abstract 654: Isoleukotoxin Promotes Autophagy and Reduces Vascular Cell Adhesion Molecule-1 Expression in Endothelial Cells
Authors: Chu Ling-yun , Cheng Hsiao-Ling , Wu Kenneth K
Published: March 2019

Rapamycin is a widely used autophagy inducer and has been shown to suppress development of atherosclerosis in animal models. However, the side effect is severe including hyperlipidemia. We identified isoleukotoxin, a metabolite of linoleic acid, as an autophagy inducer and hypothesized that it can be used to reduce inflammation of endothelial cells. To test this hypothesis we treated human umbilical vein endothelial cells (HUVECs) with tumor necrosis factor α (TNFα) along with isoleukotoxin. Rapamycin was used as a positive control to induce autophagy. Isoleukotoxin promotes autophagic flux in HUVECs as well as rapamycin. Isoleukotoxin does not inhibit activity of mammalian target of rapamycin complex, while it inhibits activity of the downstream kinase, S6K. Isoleukotoxin modulates TNFα-induced cell adhesion molecules expression in HUVECs with a similar pattern as rapamycin. In this pattern, only vascular cell adhesion molecule-1 (VCAM-1) expression is reduced but not intercellular cell adhesion molecule-1 (ICAM-1). More interestingly, time-course analysis shows that VCAM-1 expression is only reduced after 20 hours treatment, which is different from that of inhibitors of VCAM-1 transcription such as SB202190. By qPCR we confirmed that isoleukotoxin does not inhibit VCAM-1 transcription. To determine whether isoleukotoxin reduces VCAM-1 expression by inhibiting S6K-mediated protein translation, we compared the effect of isoleukotoxin with S6K siRNA. S6K siRNA reduces expression of VCAM-1 slightly and also ICAM-1, which is different from that of isoleukotoxin. After excluding the regulation of transcription and translation, we hypothesize that isoleukotoxin reduces VCAM-1 expression by promoting VCAM-1 degradation via autophagy. This hypothesis is supported by blocking of autophagy and endocytosis. Blocking of autophagy by Atg5 siRNA enhances VCAM-1 expression, suggesting that VCAM-1 may be degraded by autophagy. Blocking of endocytosis by dynasore blocks the effect of isoleukotoxin on VCAM-1 expression, suggesting that VCAM-1 is degraded after endocytosis. Here we propose that isoleukotoxin is an anti-inflammation and a potential therapeutic molecule for inflammatory vascular diseases such as atherosclerosis.
Abstract 668: Myeloid Pfkfb3 Deficiency Protect Mice From Hypoxia Induced Pulmonary Hypertension
Authors: Wang Lina , Cao Yapeng , Yang Qiuhua , Xu Jiean , Liu Zhiping , Huo Yuqing
Published: March 2019

Background:
Pulmonary hypertension is a progressive disease and perivascular inflammation is found in all types of pulmonary hypertension patients. Macrophage are innate inflammatory cells and the number of macrophages are significantly increased in the lungs of PAH patients. Glycolysis is the main pathway to generate energy and intermediate products for macrophage activation. However, the effect of macrophage glycolysis on the development of pulmonary hypertension remains unknown. As PFKFB3 is a critical regulator in glycolysis, we investigated the effect of macrophage glycolysis on pulmonary hypertension using myeloid PFKFB3 specific deficiency mice and hypoxia induced pulmonary hypertension model.

Results:
In mouse and rat hypoxia induced pulmonary hypertension models, we found that PFKFB3 was upregulated in macrophages. PFKFB3 myeloid specific depletion protected mice from the formation of pulmonary hypertension. Knockdown of PFKFB3 in macrophages suppressed the expression of growth factors and inflammatory cytokines in whole lung tissue. Also, we found that PFKFB3 knockdown decreased the numbers of perivascular macrophages and interstitial macrophages using immunostaining and FACS. The number of alveolar macrophages showed no significant difference, while the expression of growth factors and inflammatory cytokines were decreased in alveolar macrophage when PFKFB3 was depleted.

Conclusions:
PFKFB3 depletion in macrophage decreases the number of perivascular macrophages and interstitial macrophages in the lung, and suppresses the expression of growth factors and inflammatory cytokines in alveolar macrophage, thus protecting mice from the formation of pulmonary hypertension.
Abstract 669: Apabetalone (rvx-208) Decreases Risk of Major Adverse Cardiovascular Events in Diabetes Mellitus Patients With Cvd by Attenuating Monocyte Adhesion to Endothelial Cells
Authors: Tsujikawa Laura , Kulikowski Ewelina , Calosing Cyrus , Wasiak Sylwia , Gilham Dean , Halliday Christopher , Johansson Jan , Sweeney Mike , Wong Norman
Published: March 2019

Apabetalone (RVX-208, 200 mg/d) given orally to patients with diabetes mellitus (DM) and CVD leads to a 57% relative risk reduction in major adverse cardiovascular events (MACE). Potential actions of RVX-208, an inhibitor (BETi) of bromodomain extra-terminal (BET) proteins that are epigenetic readers of acetylated lysine residues within histones, in lowering MACE is explored by testing its effect on genes mediating monocyte adhesion to endothelial cells in response to high glucose (HG) and the dietary metabolite trimethyl-amine oxide (TMAO). Cultured THP-1 monocytes, HUVEC endothelial cells and primary human hepatocytes (PHH) were exposed to varying concentrations of glucose and TMAO. Results showed that HG (25.6 mM) induced Very Late Antigen-4 (VLA-4) mRNA, a gene mediating THP-1 adhesion by 1.3-fold and RVX-208 suppressed it >50%. Similarly, BETi blocked TMAO induction of VLA-4 mRNA by >50% in THP-1. In HUVECs RVX-208 abrogated HG induction of E-selectin and MYD88 mRNA by 2- and 1.3-fold, respectively and lowered TMAO induction of these mRNAs by >50%. Microbiome processing of dietary phospholipids followed by hepatic flavin mono-oxygenase-3 (FMO3) metabolism yields TMAO. In PHH exposed for 24 hrs to RVX-208, FMO3 mRNA was lower by 40% but it also suppressed a transcriptional regulator of FMO3, farnesoid X receptor (FXR). BETi suppressed both FXR mRNA and protein within 6 hrs by >80% suggesting a direct effect of BETi on the gene encoding FXR. Furthermore, ChiP data showed that BRD4, a BET protein, dissociated immediately from FXR gene upon exposure to RVX-208. Since BRD4 guides a complex containing RNA pol II along actively transcribed genes containing histones that are highly acetylated, the dissociation of BRD4 from FXR DNA would halt transcription of this gene. In summary, Apabetalone inhibits HG and TMAO enhanced adhesion of THP-1 to HUVECs a process that mimics a step in the pathogenesis of CVD. RVX-208 suppresses genes underlying cellular adhesion; VLA-4 in THP-1 and both E-selectin plus MYD88 in HUVECs. BETi blocks not only activity of TMAO but also its production by inhibiting FXR expression, a regulator of FMO3 gene transcription. The rapid actions of BETi in dissociating BRD4 from FXR DNA suggests a direct effect of RVX-208 on transcription of this gene.
Abstract 675: Biomechanical Alterations of Endothelial Cells That Affect Infection by
Listeria Monocytogenes
Authors: Bastounis Effie , Yeh Yi-Ting , Theriot Julie
Published: March 2019

Endothelial cells (ECs) line the inner lumen of vessels forming a protective barrier against spread of a variety of insults, including bacterial infections. ECs display remarkable phenotypic heterogeneity in part due to the different mechanical stimuli transmitted from their extracellular environment, including variations in subendothelial stiffness and shear flow. Although EC response to mechanical signals has been studied extensively, how bacterial uptake occurs in ECs residing on varying stiffness environments was hitherto largely unknown.

We exploited
Listeria monocytogenes
(Lm), an intracellular bacterial pathogen, to determine the biophysical relationships between ECs, their subendothelial stiffness matrix and bacterial infection. We found that adhesion of Lm onto ECs increases with increasing subendothelial stiffness in a focal adhesion kinase (FAK)-dependent manner and that FAK activity is critical for uptake of Lm by ECs. We also found that the amount of vimentin exposed at the apical surface of host ECs is reduced when FAK activity is decreased, which leads to reduced
Lm
uptake by ECs.

Our results identify a novel pathway wherein increased subendothelial stiffness sensed by ECs leads to enhanced FAK activity and host surface vimentin, that in turn increases Lm uptake. We envision that by uncovering the molecular details of vascular infection and gaining mechanistic insight into how mechanics affect infection of the vasculature, we could lay important groundwork for the development of novel therapeutic interventions against bacterial infections.
Abstract 677: Genetic Depletion of the Long Non-coding RNA H19 in Mice Protects from Elastase-induced Abdominal Aortic Aneurysms
Authors: Chernogubova Ekaterina , Busch Albert , Li Yuhuang , Jin Hong , Winski Greg , Hofmann Patrick , Boon Reinier A , Bäcklund Alexandra , Maegdefessel Lars
Published: March 2019

Long noncoding RNAs (lncRNAs) have been shown as crucial molecular regulators in various biological processes and diseases. Recently we demonstrated that lncRNA H19 is highly upregulated during abdominal aortic aneurysm (AAA) development and progression in murine models (Angiotensin II in ApoE-/- mice; porcine pancreatic elastase model (PPE) in C57BL/6 mice). Experimental H19 knock-down using specific antisense LNA oligonucleotides showed a significant reduction in AAA growth in both models. Aim of this current study was to utilize genetically mutated H19-depleted mice (H19-/-) vs. wildtype littermate controls, to assess their behavior upon experimental AAA induction using PPE. In addition, we studied the proliferation rates of smooth muscle cells, originating from either H19-/- or H19+/+ mice in a kinetic live-cell imaging system. H19-/- on a C57BL/6J background were exposed to PPE. The aortic diameter in H19-/- mice was compared to WT littermate controls (upon PPE-AAA induction) at baseline, and then consecutively at days 7, 14, and 28. Primary mouse aortic smooth muscle cells were isolated from wild type or H19-depleted aortas, and cultured and monitored in the IncuCyte live cell imaging system for 48 hours, in an effort to study their proliferation rate. H19-/- mice upon PPE-AAA induction displayed significantly lower diameters throughout the study compared to WT controls. Primary aortic smooth muscle cells from H19-depleted mice showed greatly increased proliferation rates (based on cell confluency detection) in our kinetic live-cell imaging system in comparison to WT control cells. In conclusion, our study in H19-depleted mice supports our previously presented efforts, that H19 is an important contributor to experimental AAA development and progression. Further mechanistic studies will have to reveal the molecular properties of this long non-coding RNA in smooth muscle cell survival and proliferation.