bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 386: Dietary Oxidized Linoleic Acid Modulates PCSK9 and Associated Genes
Authors: Ochin Chinedu , Garelnabi Mahdi O
Published: March 2019

Introduction:
We have previously shown that oxidized linoleic acid (OxLA) modulate plasma lipids. In this study we investigated the role of dietary OxLA on PCSK-9 and its associated genes.

Method:
Liver tissue from four groups of normal C57BL6 mice were feed; plain chow, chow supplemented with linoleic acid (9mg), chow supplemented with a low concentration of OxLA (9mg) and chow supplemented with a high concentration of OxLA (18mg) were analyzed for PCSK-9 and related genes. RNA from these livers samples was extracted with Trizol, assessed for purity by agarose gel electrophoresis and quantified using fluorometry. cDNA was synthesized from total extracted RNA and subsequently used for qRT-PCR analysis. Gene expressions of
PCSK-9, SREBP-1, HNF-1α
and
PPARα
were measured and normalized to
GAPDH
.
Total, free
and
cholesterol ester
levels in the liver were analyzed spectrophotometrically. Experiments were performed in triplicates and the difference between means was determined using a student’s T-test with a p value of <0.05 accepted as statistically significant.

Results:
PCSK-9 gene expression was markedly elevated in the mice fed high concentration OxLA, SREBP-1 gene levels in these of mice was also significantly elevated in comparison to the other 3 groups which had relatively the same levels of SREBP-1 gene expression. Conversely HNF-1α gene expression levels were elevated in all groups except the mice on low concentration OxLA. PPARα gene levels were significantly down regulated in the mouse on high concentration OxLA. Plasma total cholesterol levels were significantly elevated in both groups on OxLA.

Conclusion:
Elevated cholesterol levels in the livers of mice fed high concentration OxLA was accompanied with increased PCSK-9 expression. These results show that OxLA modulate PCSK-9 and its associated genes expression and promote accumulation of liver cholesterol.
Abstract 397: Cotinine, but Not Nicotine, Increased ApoA-I Mediated HDL Synthesis in THP-1 Cells
Authors: Tsujita Maki , Narayanaswami Vasanthy
Published: March 2019

Aim:
Cotinine is known as a stable metabolite of nicotine, the plasma half-life is over 20-30 hrs. Here, we evaluated its characteristic properties on the apoA-I mediated HDL generation.

Method:
Cotinine (5.6 to 11.2μM), or nicotine (5 to 150μM) were supplemented to medium and co-incubated with apoA-I (10μg/mL) for 18 hrs with THP-1 cells to determine their effect on efflux activity. Cellular ACAT accessible cholesterol pool size was determined by [
14
C]oleic acid labeling of cells for 60 min immediately after removal of the culture medium. The culture medium and the cellular lipids were extracted by organic solvents and cholesterol and phospholipids content were determined. Nicotine was administered orally as an aqueous solution (10 μM) to C57BL/6 mice for 10 days and the plasma lipoprotein profiles were measured by gel-HPLC (tandem TSK-lipopropak
XL
) with online enzymatic lipid assay system (Skylight biotech, Inc.).

Results:
Supplementation of nicotine into the medium showed no effect on the apoA-I mediated cellular cholesterol release from THP-1 cells. On the other hand, cotinine increased apoA-I mediated cellular cholesterol efflux 2.8 fold. ACAT accessible cholesterol pool size was dramatically decreased to 80% and 58% of control with 5 and 10 μM cotinine treatment, respectively, indicating that cotinine effectively increased apoA-I/ABCA1 mediated cellular cholesterol efflux. Mice with oral nicotine administration showed increased plasma HDL by 11.4% (50.1±4.4 and 55.8±3.9 mg/dL for control and nicotine treatment, respectively).

Conclusions:
Cotinine increased apoA-I mediated cellular cholesterol efflux in THP-1 cells. The cellular ACAT accessible cholesterol pool size reduction is one of the early reactions of apoA-I/ABCA1 mediated cellular cholesterol efflux. Cotinine seemingly modifies cellular factor/factors to induce a change in cholesterol distribution. Mice administrated oral nicotine may convert the compound to cotinine by CYP2A6 and aldehyde oxidase activities in their liver, and which likely increased HDL generation. Further experiments are necessary to examine the direct effect of cotinine on the ABCA1 transporter for increasing HDL assembly system.
Abstract 411: Human Species-specific Loss of the CMP-N-acetylneuraminic Acid Hydroxylase Fuels Atherosclerosis Development via Intrinsic and Extrinsic Mechanisms
Authors: Kawanishi Kunio , Ramms Bastian , Diaz Sandra , Do Raymond , Varki Nissi , Varki Ajit , Gordts Philip
Published: March 2019

Cardiovascular disease (CVD) events due to atherosclerosis are very common in humans, but rarely occur spontaneously in other mammals, absent experimental manipulation. All humans exhibit a species-specific deficiency of the common mammalian sialic acid N-glycolylneuraminic acid (Neu5Gc), due to pseudogenization of the CMP-N-acetylneuraminic acid (Neu5Ac) hydroxylase (CMAH) gene, which occurred in hominin ancestors about 2-3 million years ago. Human-like Cmah
-/-
mice that express only the precursor sialic acid Neu5Ac are more prone to insulin resistance and have more reactive macrophages and T cells. Human dietary consumption of Neu5Gc (primarily from red meat), can acts as a foreign “xenoantigen” in humans that gets metabolically incorporated into endogenous glycoproteins. Humans with circulating anti-Neu5Gc “xeno-autoantibodies” can thus potentially develop local chronic inflammation or “xenosialitis” at sites of Neu5Gc accumulation such as endothelial cells and in atherosclerotic plaques. In this study we set out to test if human CMAH deficiency contributes to CVD via multiple intrinsic and extrinsic mechanisms. Cmah
-/-
Ldlr
-/-
mice had increased atherogenesis on a Neu5Gc-free high fat diet (HFD), compared to Cmah
+/+
Ldlr
-/-
mice. This was not associated with cytokine levels in plasma, but increased cytokine expression was seen in Cmah
-/-
Ldlr
-/-
macrophages in comparison to Cmah
+/+
Ldlr
-/-
. The baseline relative hyperglycemia of the Cmah
-/-
Ldlr
-/-
mice was also enhanced on a Neu5Gc-free HFD. When such mice were immunized to develop human-like levels of anti-Neu5Gc antibodies, they had a 2.5-fold increase in atherosclerosis on a Neu5Gc-rich HFD compared to control Neu5Ac-rich or sialic acid-free HFD feeding. Drastically advanced lesions with increased necrotic core areas and infiltration of macrophages and T-cells accompanied increases in atherosclerotic area and lesion volume. None of these differences were explained by changes in lipoprotein profiles or insulin sensing. Human evolutionary loss of CMAH likely contributes to atherosclerosis propensity, via both intrinsic mechanisms such as amplified chronic inflammatory response and hyperglycemia; and extrinsic mechanisms such as red meat-derived Neu5Gc-induced xenosialitis.
Abstract 415: HDL-small RNA Gene Regulatory Networks Alter T Cell Signalling
Authors: Michell Danielle L , Zhao Shilin , Sheng Quanhu , Ormseth Michelle J , Stein C. Michael , Major Amy S , Vickers Kasey C
Published: March 2019

Small non-coding RNAs (sRNA) are critical regulators of adaptive immunity and the flow of information between immune cells likely extends beyond cytokines and danger signals to sRNAs. microRNAs (miRNA) are sRNAs that post-transcriptionally regulate gene expression and are transferred between cells within intercellular communication networks. In addition to miRNAs, another class of sRNAs has emerged as critical regulators of gene expression - tRNA-derived small RNAs (tDRs). During cell stress, parent tRNAs are cleaved by RNaseIII enzymes, e.g. Angiogenin (ANG), and tDRs suppresses protein synthesis and gene expression; however, the functional relevance of ANG and tDRs in T cell activation is unknown. To determine if ANG is altered during T cell activation, ANG expression was quantified in anti-CD3 and anti-CD28 activated human CD4+ T cells, and we found that both mRNA and protein levels were significantly (
p
<0.05) increased compared to non-activated T cells by real-time PCR and ELISA, respectively. Strikingly, 12 T cell tDRs were significantly upregulated (
p
<0.05) during activation, as quantified by high-throughput sRNA sequencing (sRNA-seq). In addition, tDR-GlyGCC was found to be readily exported from CD4+ T cells to HDL after 24 hr post-activation. Moreover, sRNA-seq analysis showed that tDRs (e.g. tDR-GlyGCC) are highly abundant on human HDL. Based on these results, we hypothesized that HDL mediates T cell-originating tDR intercellular communication networks. In support, we found that HDL transferred tDR-GlyGCC from T cells to monocytes, as evidenced by an 18-fold increase (
p
<0.05) in tDR-GlyGCC in recipient cells. In conclusion, we demonstrate that HDL facilitates a novel intercellular communication network of tDRs between immune cells which is likely enhanced during T cell activation.
Abstract 422: Association of Remnant Lipoprotein Cholesterol and Levels of Glucose and Insulin: the Very Large Database of Lipids
Authors: Quispe Renato , Baradaran Noveiry Behnoud , Faridi Kamil F , Martin Seth S , Toth Peter P , Jones Steven R
Published: March 2019

Background:
A defining feature of diabetic dyslipidemia is elevation of triglyceride-rich lipoproteins, particularly remnant lipoprotein cholesterol (RLP-C). Lowering these lipoproteins may reduce risk of atherosclerotic cardiovascular disease in diabetic patients, though the relationship between RLP-C and hyperglycemia as well as hyperinsulinemia remains inadequately characterized. The purpose of this study was to determine the association of RLP-C with serum glucose and insulin across a broad range of insulin resistance.

Methods:
We used a sample of individuals from the Very Large Database of Lipids with measured fasting serum glucose and insulin levels, as well as lipoprotein cholesterol levels measured by the Vertical Auto Profile test (Atherotech, Birmingham, AL). RLP-C was defined as the sum of VLDL
3
-C + IDL-C. The study population was divided into deciles of HOMA-IR, calculated as [Insulin х Glucose/405]. We performed multivariable linear regression models to determine associations of RLP-C with insulin and glucose after adjusting for age, sex, real LDL-C, triglycerides, AST, BUN and creatinine. Covariates not normally distributed were log-transformed. Analysis was performed in overall population and across HOMA-IR deciles.

Results:
We included a total of 146,826 individuals (43.6% male, mean age 54.9 ± 15.9 years). Median values were: insulin, 9 uU/mL; glucose, 95 mg/dL; RLP-C, 26 mg/dL. The models in our Table explained 60% of variance in RLP-C. Overall, insulin (β=-1.85, p<0.001) and glucose (β=-0.84, p<0.001) had significant negative associations with RLP-C. However, levels of RLP-C were significantly associated with insulin but not glucose across most HOMA-IR deciles (Table).

Conclusion:
RLP-C is significantly associated with levels of serum insulin but not with glucose across a spectrum of insulin resistance. Further characterization of the relationship between RLP-C and serum insulin is needed to help guide future therapies for patients with diabetes.
Abstract 426: Atherosclerotic Risk Factors Correlate with Carotid Intimal Media Thickness in Participants of the Childhood Obesity Study in South Texas: Preliminary Results
Authors: Karere Genesio , Bishop Andrew , Cox Laura , Mercado-Deane Maria-Gisera , Cuda Suzanne
Published: March 2019

Rationale:
The prevalence of childhood and adolescent obesity is increasing in the United States, where one-third of children and adolescents are affected. This is especially true in the Hispanic population. It is well known that obesity is positively associated with increased atherosclerosis, the primary cause of cardiovascular disease (CVD).

Our study objective was to determine whether carotid intima-media thickness (CIMT) is associated with the severity of obesity and cardiovascular risk factors in obese Hispanic adolescents in San Antonio, Texas. We present for the first time, preliminary results for an on-going, Childhood Obesity Study at The Children’s Hospital of San Antonio, where obese children in South Texas are referred for weight management.

Methods:
We present data for 36 obese subjects (males= 21) with an average % of the CDC 95
th
BMI percentile of 139 at baseline (first clinic visit). The mean age was 15
+
1 years. We assessed cardiovascular risk factors and measured CIMT using B-mode ultrasonography on the left and right carotid arteries. For each patient, we calculated mean CIMT for carotid internal artery, common carotid artery, and carotid bulb, and the mean for all the CIMT measurements.

Results:
There were no age-sex differences in BMI, plasma LDL-C, high sensitivity C-reactive protein (hs-CRP) and CIMT. The mean CIMT for the common carotid artery (CIMT-CCA), carotid internal artery (CIMT-CIA), and carotid bulb (CIMT-CB) were 0.50±0.12, 0.43±0.22, and 0.60±0.29 mm respectively. BMI correlated with CIMT-CIA (p= 0.012) and CIMT-CB (p= 0.002). The correlation remained significant with the means of all the CIMT measurements (p= 0.002). Plasma hs-CRP and LDL-C concentrations correlated with CIMT-CIA (p= 0.045) and the means for all CIMT measurements (p= 0.027). We did not observe significant correlations with CIMT for the common carotid artery.

Conclusion:
Cardiovascular risk factors assessed in adolescents who are obese correlate with CIMT, a surrogate for atherosclerotic burden, suggesting that these obese adolescents have early onset atherosclerosis. Our results are preliminary and should be interpreted cautiously.
Abstract 431: Sex Differences in Manifestation of Subclinical Coronary Atherosclerosis: Analysis of Lipoprotein Sub-Fractions in Healthy but High Risk Individuals
Authors: Veledar Emir , Saxena Anshul , Arias Lara , Aneni Ehimen , Garelnabi Mahdi , Nasir Khurram
Published: March 2019

Background:
Lipoproteins-rich triglycerides are associated with markers of coronary atherosclerosis such as coronary artery calcification (CAC) in young high-risk population. This study examines differences between sexes associated with lipoprotein sub-fractions and atherosclerosis.

Methods:
The study presents analysis from baseline data of a randomized trial of employees of Baptist Health South Florida with metabolic syndrome or diabetes. Participants completed lipoprotein sub-fraction analysis at baseline using ion mobility technique. Subjects above 35 years had a CAC test completed. We tested for differences between correlational matrices of the original variables. The same analysis was repeated for sets of 3 principal components (PC) that were computed for the combination of all lipoprotein subclasses.

Results:
The study population (N=170) was largely female (128) with a mean age of 58 years. Three PCs accounted for 83% variation in the sample. Distribution of atherosclerostic PC2 by sex was entirely different between sexes (Wishart test p=.004). In addition, a significant Jennrich’s test (p < .0045) implied that the inter-correlations among all dependent variables for different groups were dissimilar; with the finding of significant angles between PC components by sex. PC2, with main contributions from VLDL particles, was in the positive direction; however PC3 with large LDL particles was in the negative direction. Atherogenic lipoprotein profiles were significant only for males, with PC keeping their independent predictability.

Conclusion:
In a relatively young high-risk population, there were sex-specific differences in lipoprotein sub-fractions profiles that are associated with increased risk of atherosclerosis. The relationship was much weaker for males compared to females (see graph). These findings points towards a possible sex-linked lipoprotein sub-fraction association with the atherosclerosis pathogenesis.
Abstract 432: Differential MicroRNA Expression in Subcutaneous Abdominal and Lower-body Fat Depots
Authors: Evans Jared , Sun Zhifu , Covassin Naima , Sert-Kuniyoshi Fatima H , Somers Virend K , Singh Prachi
Published: March 2019

Background:
Abdominal fat elevates while lower-body (LB) fat diminishes cardiovascular risk. However, molecular mechanisms differentiating the two fat depots are not completely understood. MicroRNA (miRNA) are important regulators of gene expression with widespread effects on several proteins.

Objective:
To explore miRNA differences between the two fat depots using unbiased next-generation sequencing (NGS) approach.

Methods:
Total RNA isolated from paired subcutaneous (sc) fat tissue obtained from abdomen (ABD) and LB (femoral) region of 12 healthy subjects (7 male; age: 27 ± 5 years; BMI: 23.5 ± 3.2 kg/m
2
) was used to quantify miRNA expression. Body fat distribution was determined using dual-energy X-ray absorptiometry and abdominal computed tomography scans. RNA was prepped using the NEBNext small RNA library prep kit and sequenced using Illumina HiSeq 2000. Raw sequence data were processed using CAP-miRSeq pipeline. miRNA expression between the two depots was evaluated using EdgeR. Relationship between miRNA and regional fat depots were examined using Spearman’s correlation. Adjusted or unadjusted P value <0.05 was considered significant.

Results:
Using NGS, 383 ± 55 known miRNA with ≥5 raw reads were detected in the study samples. Principal component analysis including all miRNAs showed no any clear separation between ABD or LB fat tissue. However, 37 miRNAs were found to have altered expression in the two depots. miR-196a-5p (log2FC=1.138, p<0.001), miR-146b-5p ( log2FC=1.388, p<0.001), miR-1287 ( log2FC=1.059, p<0.001), miR-1247-5p (1.002, p<0.001), and miR-346 (log2FC =-1.240, p=0.002) were among the top five differentially expressed miRNA in ABD Vs. LB fat. Interestingly, ABD fat tissue expression of miR196a-5p (rho=-0.58, p=0.047) and miR146b-5p (rho=0.76, p=0.004) correlated with visceral fat while miR146b-5p (rho=0.66, p=0.02) and miR1287 (rho=0.79, p=0.002) correlated with ABD sc fat.

Conclusions:
Several miRNAs are differentially expressed between ABD and LB fat tissue which may partly contribute to depot specific alterations in adipose tissue function and consequent cardiometabolic risk. Furthermore, the associations between miRNA and regional fat depots suggest a functional role of these miRNA in fat distribution.
Abstract 436: Talin1-Dependent Integrin Activation Drives Endothelial Inflammation and Fibronectin Deposition
Authors: Al-Yafeai Zaki , Yurdagul Arif , Petrich Brian , Orr A. Wayne , Orr A. Wayne
Published: March 2019

Integrin activation plays a critical role in regulating leukocyte and platelet adhesion, but less is known concerning integrin activation in adherent cells. In endothelial cells (ECs), both shear stress and oxidized LDL (oxLDL) promote integrin activation, and blocking the interactions between integrins and provisional matrix proteins, such as fibronectin, limits proinflammatory responses. However, current studies fail to demonstrate a causal role for integrin activation in this response due to the inability to specifically uncouple integrin activation while maintaining integrin function in cell adhesion. Therefore, we isolated ECs from mice containing an L325R mutation in talin1, the classic mediator of integrin activation, that selectively inhibits talin1-induced integrin activation while preserving its ability to link integrins to the actin cytoskeleton. We now show that talin1 L325R ECs show slowed adhesion and spreading, but cells eventually adhere, spread, and form focal adhesions. In addition, talin1 L325R ECs lack shear stress and oxLDL-induced α5β1 integrin activation, but basal levels of integrin activation remain similar between wildtype and Talin1 L325R ECs. Additionally, shear stress does not induce NF-κB activation or proinflammatory gene expression in talin1 L325R ECs. Consistent with abrogating inflammation, talin1 L325R completely blunts shear stress-induced fibronectin fibrillogenesis, a main initiating event in atherosclerosis development, through mechanisms involve blunting translocation of tensin1- α5β1 to fibrillar adhesions, the primary machinery of fibronectin assembly. Similarly, talin1 L325R ECs blocks endothelial inflammation and fibronectin deposition in response to oxLDL. However, preventing talin1-dependent integrin activation does not block all shear stress-mediated signaling, and proinflammatory responses to cytokines and LPS remain unchanged. These data suggest a model whereby flow and oxLDL mediate talin1-induced α5β1 integrin activation, which is required for flow and oxLDL-induced inflammation and fibronectin assembly.
Abstract 439: Statins Disrupt Rac Regulation Leading to Increased Atherosclerotic Calcification
Authors: Healy Abigail , Mantsounga Chris , Berus Joshua M , Christensen Jared , Watt Jerome , Ceneri Nicole , Nilson Rachael , Neverson Jade , Choudhary Gaurav , Morrison Alan
Published: March 2019

Coronary artery disease caused by atherosclerosis is the leading cause of morbidity and mortality in the world. Calcification of atherosclerotic plaque is predictive of increased cardiovascular events; however, recent studies demonstrate that increased calcium density may be associated with more stable disease. Therapeutic modulation of calcified atherosclerotic plaque composition may have potential to reduce event risk. HMG-CoA reductase inhibitors (statins) reduce the risk of cardiovascular events. Moreover, recent data demonstrate that statin use is associated with increased plaque calcification, yet the underlying mechanisms for this process are not well understood. Our laboratory identified a Rac-dependent pathway that promotes IL1B-driven atherosclerotic calcification. Here, we hypothesize that statins disrupt isoprenylation of Rac1, resulting in increased activation of Rac1 and consequent IL1B expression. Results demonstrated statin-induced loss of Rac1 isoprenylation disrupted a complex between Rac1 and its inhibitor, Rho-GDI, in primary macrophages. This led to increased GTP-bound (activated) Rac1 in a statin dose-dependent manner. This phenomena was rescued by a precursor isoprenyl group for Rac1. Statin-treated primary macrophages expressed higher IL1B mRNA and secreted more IL1B protein in response to inflammasome activation. Small molecule inhibition of Rac1 and
Rac1
gene-deletion mitigated this statin-induced effect.
ApoE

-/-

mice placed on high fat diet with statin therapy revealed increased plaque calcification in association with elevated serum IL1B protein. In summary, we have defined a potential mechanism of statin-induced calcification, whereby statins disrupt Rho-GDI-mediated inhibition of Rac1.
Abstract 446: Ampk Regulates Endothelial Mirna Biogenesis Through Nucleolin
Authors: Gongol Brendan , Marin Traci , Shyy John
Published: March 2019

Background:
A characteristic of atherosclerosis is the focal distribution of lesions in the arterial tree despite the systemic nature of cardiovascular impairments such as hyperlipidemia. While pulsatile shear stress (PS, atheroprotective) maintains endothelial homeostasis, oscillatory shear stress (OS, atherogenic) causes endothelial dysfunction. Previously, we and others have identified AMP activated protein kinase (AMPK) is a master regulator in vascular endothelial cells (ECs) in response to PS. To further decipher the atheroprotective effect of AMPK, bioinformatic approaches identified a broad array of AMPK-regulated pathways that regulate cellular functions beyond its canonical catabolic regulation. Among other newly identified substrates, nucleolin (NCL) is a multifunctional protein and the newly identified AMPK phosphorylation of NCL S328 may be a major mechanotransduction event hindering miRNA biogenesis in ECs.

Methods and Results:
Using AMPK kinase assays and phospho-S328 NCL antibody, we identify that the PS-activated AMPK increased phospho-S328 NCL. Cellular fractionation and immunofluorescence indicated NCL localization throughout the nucleus and cytoplasm which was sequestered to the nucleus following AMPK activation. This indicated bipartite NCL functions, acting as a chaperone under dephospho-S328 conditions and maintaining a transcriptional function upon phosphorylation. Bioinformatics analysis validated by RNA immunoprecipitation showed that the OS-induced miR-93 and miR-484 were novel NCL-regulated miRNAs. Putative miR-93 and miR-484 targets, including eNOS and BMPR2 were cross referenced to RNA-seq data from ECs exposed to PS and OS. ECs transfected with anti-miR-93 and anti-miR-484 exhibited ratified NO bioavailability and attenuated inflammation. In response to PS stimulation, however, AMPK activation increased NCL phosphorylation resulting in its binding to the KLF2 promoter to induce KLF2 that increased NO bioavailability and inhibited miR-93 and miR-484 induction.

Conclusions:
These results demonstrate that AMPK can inhibit the expression of atherogenic miR-93 and miR-484 in ECs through AMPK phosphorylation of NCL S328.
Abstract 448: Stress Granule Formation as a Molecular Indicator of Atherosclerotic and Vascular Disease Progression
Authors: Herman Allison B , Vrakas Christine N , Ray Mitali , Kelemen Sheri E , Autieri Michael V
Published: March 2019

The molecular mechanisms by which atherogenic stress contributes to the pathophysiology of disease remain to be fully characterized. RNA-binding proteins (RBPs) regulate vascular cell (VSMCs and ECs) response to inflammatory stimuli by fine-tuning mRNA stability, and protein abundance. RBPs such as HuR and FXR1 regulate mRNA transcript abundance by binding AU-rich elements (AREs) in the 3’UTR permitting the transcript stabilization or destabilization, respectively. Most inflammatory transcripts including TNFα contain conserved AREs in their 3’UTR, and inflammatory stimuli activate RBPs to alter the landscape of inflammatory mediators. This post-transcriptional regulation of inflammatory transcripts by RBPs islocalized to structures called stress granules (SGs), which are mRNA-protein assemblies formed from non-translating mRNAs, ribosomal subunits, among other components. The manner by which these complexes form between mRNA and RBPs (mRNPs) under inflammatory or pathological conditions remains unknown, particularly with respect to vascular inflammatory disease. We hypothesize that the presence and quantity of stress granules are a critical cellular response to atherogenic stimuli. We identified SGs in cultured VSMCs and ECs treated with clotrimazole, a known inducer of SGs by co-staining for PABP, a SG marker, along with RBPs HuR and FXR1, both known to co-localize to SGs. VSMC treated with clotrimazole had increased eIF2α phosphorylation. LDLR-/- mice fed high fat diet for 14 weeks also demonstrated significantly increased PABP expression in the smooth muscle and endothelial cells of the aortic arch as compared to WT control. Atherogenic and hypoxic stimuli induced the formation of SGs in cultured primary human VSMCs and ECs. Interestingly, we found VSMCs pre-treated with anti-inflammatory cytokine IL-19 followed by clotrimazole significantly reduced the formation of SGs per cell in a time dependent manner (n=50, p<0.05) as well as reduction in PABP expression, suggesting anti-inflammatory treatment effects SG formation. Taken together, these results have led to the hypothesis that SG formation in atherosclerosis is driven by inflammatory stimuli, and that anti-inflammatory treatment may lessen atherosclerosis progression and plaque formation by reduction of SGs.
Abstract 451: Radiation Induced Atherosclerosis on Mice
Authors: Ko Kyung-Ae , Kotla Sivareddy , Wang Yin , Fujii Yuka , Vu Hang T , Vankatesulu Bhanu P , Thoman Tamlyn N , Medina Jan , Gi Young Jin , Hada Megumi , Grande-Allen Jane , Patel Zarana S , Milgrom Sarah A , Kirshnan Sunil , Fujiwara Keigi , Abe Jun-ichi
Published: March 2019

Rationale:
The high prevalence of cardiovascular disease (CVD) in cancer survivors after ionizing radiation (IR) therapy has long been recognized, but the mechanism for this is unknown. Previous studies reported that the plaques in IR therapy survivors exhibit characteristics of increased instability without changes in the plaque size.

Objective:
To study the mechanism of IR induced CVD in humans, we decided to develop an IR mouse model that mimics human CV events.

Methods and Results:
We fed
LDLR-/-
mice or C57 mice with a single injection of adeno -associated virus vector(AAV) encoding a gain-of function of PCSK9 with a high fat diet, then irradiated the mice with different doses of IR (2 Gy, 5 Gy, 10 Gy). Whole body radiation was given 2 Gy, while localized IR to the neck and thoracic area was given at 5 Gy (1 time) or 10Gy (weekly 5 Gy, twice).About 2~3 weeks after the IR treatment, we performed left partial carotid artery ligation (PCL), and 3 weeks later examined the extent of plaque formation and cardiac abnormality by comparing those detected in non-irradiated control mice. The lesion area ratio between the partially ligated left carotid artery (LCA) and the right carotid artery (RCA) was significantly increased in the IR-treated group compared to the non-IR-treated group (3.1 ± 0.3 vs.1.8 ± 0.4, n = 7-8, p = 0.03). In addition, the lesion in the IR-treated of mice had significantly larger necrotic cores with a thinner fibrous cap when compared to those in non-irradiate mice. Furthermore, IR-treated mice showed cardiac hypertrophy, in which we found a significant increase of the wall thickness of mid-to small sized arteries as well as perivascular fibrosis, which were not noticed in the non-IR-treated mice.

Conclusions:
Our results demonstrate that IR not only increases atherosclerotic and vulnerable plaque formation but also cardiac hypertrophy with mid- and micro-vascular wall swelling. The increase of mid- and micro-vascular wall thickness may explain the heart failure preserved heart failure, which is observed in the majority of heart failure patients after IR treatment.
Abstract 452: Estrogen Inhibits LDL Transcytosis by Coronary Artery Endothelial Cells via GPER and SR-BI
Authors: Ghaffari Siavash , Sugiyama Michael G , Naderi Nabi Farnoosh , Lee Warren L
Published: March 2019

Objectives:
Premenopausal women have a significantly lower rate of atherosclerosis than men, an effect attributed to estrogen. The atheroprotective effects of estrogen are independent of circulating lipid levels and may be due to an effect on the vessel wall. Early on in atherosclerosis, circulating LDL is able to cross an intact endothelial monolayer by transcytosis and accumulate in the intima. Little is known about the mechanisms of transcytosis but using novel assays we recently discovered a role for the SR-BI and ALK1 receptors. We hypothesized that estrogen can attenuate LDL transcytosis by coronary artery endothelial cells.

Approach and Results:
Using a recently described assay based on total internal reflection fluorescence microscopy, we quantified transcytosis of LDL across human coronary artery endothelial cells treated with physiological concentrations of estrogen. Estrogen significantly attenuated LDL transcytosis by endothelial cells from male donors; the effect was ligand-specific as transcytosis of albumin was not affected. Estrogen caused down-regulation of endothelial SR-BI but not ALK1 and over-expression of SR-BI was sufficient to restore LDL transcytosis. Similarly, depletion of SR-BI by siRNA attenuated endothelial LDL transcytosis and prevented any further effect of estrogen. In contrast, treatment with estrogen had no effect on SR-BI expression by liver cells from a male donor. Inhibition of estrogen receptors α and β had no effect on estrogen-mediated attenuation of LDL transcytosis. However, estrogen’s effect was blocked by depletion of the G-protein coupled estrogen receptor (GPER) by siRNA. GPER was found to be enriched in endothelial cells compared to hepatocytes and is known to signal via transactivation of the epidermal growth factor receptor (EGFR); we observed that inhibition of EGFR also prevented the effect of estrogen on LDL transcytosis. Lastly, male mice demonstrated more vascular deposition of LDL than age-matched female mice after acute injection.

Conclusions:
Physiological concentrations of estrogen significantly inhibit LDL transcytosis by down-regulating endothelial SR-BI; this effect requires GPER. This may contribute to the lower rates of atherosclerosis in premenopausal women.
Abstract 454: Repression of Map1lc3a During Atherosclerosis Progression Plays an Important Role in the Regulation of Vascular Smooth Muscle Cell Phenotype
Authors: Magné Joelle , Paloschi Valentina , Gonçalves Isabel , Saliba-Gustafsson Peter , Skogsberg Josefin , Razuvaev Anton , Jin Hong , Li Yuhuang , Ketelhuth Daniel F.J. , Maegdefessel Lars , Hedin Ulf , Eriksson Per , Ehrenborg Ewa
Published: March 2019

Background:
Autophagy is a cell survival mechanism, which has been implicated in atherogenesis in mouse models by studying core autophagy machinery proteins using knock-out models. MAP1LC3A and MAP1LC3B play a key role in autophagy activity and have been implicated as prognostic factors in several human cancers. However, data on the involvement of autophagy in human atherosclerotic disease and plaque vulnerability are still sparse and completely lacking with regards to the involvement of MAP1LC3.

Approach and Results:
Using two independent biobanks of human carotid atherosclerotic plaques, we observe that MAP1LC3A mRNA and protein levels are decreased in plaques from patients with symptomatic disease compared to asymptomatic. Notably, MAP1LC3A mRNA levels strongly correlate with vascular smooth muscle cell markers, while MAP1LC3B does not. In in vivo models, we show that MAP1LC3A mRNA is downregulated during the progression of atherosclerosis in hypercholesterolemic mice as well as upon hyperplasia induced by balloon-injury in rats. In vitro, we show that ablation of MAP1LC3A in human carotid VSMC induces a transient compensatory increase in myocardin, a master regulator of vascular smooth muscle cell phenotypic switch.

Conclusions:
Taken together, these results demonstrate that reduced MAP1LC3A expression is a relevant marker of vulnerable plaque phenotype, suggesting an impact on vascular smooth muscle cell biology in the context of atherogenesis.
Abstract 455: Inhibition of Histone Deacetylase 6 Activity Provides Protection Against Atherogenesis: a Role for HDAC6 NEDDylation
Authors: Nomura Yohei , Rossberg Max C , Bhatta Anil , Romer Lew , Berkowitz Dan , Pandey Deepesh
Published: March 2019

Rationale:
Histone Deacetylase 6 (HDAC6) regulates Cystathionine Gamma Lyase (CSEγ)- hydrogen sulfide producing enzyme known to play a critical role in endothelial function. However, a role for HDAC6 in atherogenesis is unknown.

Objective:
To determine whether pharmacological inhibition of HDAC6 inhibition by tubacin would attenuate atherogenesis and to elucidate specific molecular mechanism(s) that regulate endothelial HDAC6 activity.

Methods and Results:
We evaluated whether administration of tubacin attenuated or reversed the endothelial dysfunction and atherosclerosis induced in mice by a single i.p injection of PCSK9 AAV followed by a high fat diet (HFD) for 12 weeks. Tubacin significantly blunted PCSK9-induced increases in pulse wave velocity (index of vascular stiffness and overall vascular health) that are also seen in atherogenic mice. Furthermore, tubacin protected vessels from defective vasorelaxation, as evaluated by acetycholine-mediated relaxation using wire myograph. Plaque burden defined by Oil Red O staining was also found to be significantly less in mice that received tubacin than in those that received PCSK9 alone. Inhibition of the NEDD8 -activating enzyme 1 (NAE1) by MLN4924 significantly increased HDAC6 activity in Human Aortic Endothelial Cells (HAEC). Interestingly, levels of HDAC6 remain unchanged. This finding is consistent with the etiology of the change in function being the post-translational modification of HDAC6 by NEDD8, and not due to its overall abundance. HAEC exposed to the atherogenic stimulus OxLDL exhibited enhanced HDAC6 activity which was attenuated in a dose-response manner by pre-treatment with MLN4924.

Conclusion:
HDAC6 inhibition by tubacin and/or MLN4924 represents a novel pharmacological intervention for atherogenesis.
Abstract 457: Feedback Regulation of 3-Hydroxy-3-methylglutaryl-coenzyme a Reductase in Livers of Mice
Authors: Su Shan , Garland Kristina , Jo Youngah , Hwang Seonghwan , Young Gennipher , Fuentes Iris , Schumacher Marc , Elsabrouty Rania , Johnson Brittany , Debose-Boyd Russell A
Published: March 2019

Introduction:
HMG-CoA reductase is a membrane protein of the endoplasmic reticulum that catalyzes reduction of HMG-CoA to mevalonate, a rate-limiting step in the synthesis of cholesterol and nonsterol isoprenoids, which exert feedback control on HMGCR through multiple mechanisms. These mechanisms ensure constant synthesis of essential nonsterol isoprenoids, while avoiding toxic cholesterol accumulation. One mechanism involves sterol-induced ubiquitination of HMGCR, marking the enzyme for degradation from ER membranes, a process augmented by nonsterol isoprenoids. We examine the contribution of sterol-accelerated ubiquitination/degradation to overall regulation of HMGCR in livers of mice.

Methods:
Forty mice, including 20 wild-type (WT) and 20 knock-in (Ki) mice expressing ubiquitination-resistant HMGCR, were fed diets containing only chow, or chow supplemented with 0.1, 0.3, or 1% cholesterol. After five days of feeding, livers were harvested for measurements of cholesterol and triglycerides, immunoblot analysis, and qRT-PCR of genes related to cholesterol, nonsterol isoprenoid, and fatty acid synthesis.

Results:
Normalization of mRNA to protein levels indicates that HMGCR Ki livers contain a more HMGCR protein despite mRNA downregulation. Protein and gene expression of SREBP2 and its target genes, which contribute to cholesterol synthesis, decreased as expected with increased dietary cholesterol. Conversely, protein and gene expression of SREBP1 and its target genes increased, likely due to SREBP1c predominance toward fatty acid synthesis, which prevents cholesterol accumulation.

Conclusion:
The increase in HMGCR protein relative to mRNA suggests that significant post-transcriptional regulation exists in the form of impaired degradation. Furthermore, these normalized values indicate that accumulation of protein is primarily due to impaired degradation at lower cholesterol levels (chow, 0.1%); however, at high cholesterol levels (0.3, 1%), a greater degree of transcriptional control from sterol-mediated inhibition of SREBP2 regulates HMGCR due to negative feedback. This study demonstrates the role of degradative control on inhibition of HMGCR and may assist in reducing HMGCR accumulation during statin therapy.
Abstract 459: Protein Glutathionylation Promotes Calcification via Oxidative DNA Damage in Human Valve Endothelial Cells
Authors: Valerio Vincenza , Montanari Silvia , Songia Paola , Porro Benedetta , Moschetta Donato , Cavallotti Laura , Myasoedova Veronika , Poggio Paolo
Published: March 2019

Background:
Calcific aortic valve disease (CAVD) is one of the most common form of heart valve disease and affects 3% of the population. The initial phase, called aortic valve sclerosis (AVSc), is linked to inflammation and oxidative stress with direct consequences on endothelial cells. Thus, we aimed to evaluate the effects of protein glutathionylation caused by oxidative damage in endothelial cells.

Methods:
We analysed blood samples from 58 subjects, 29 with AVSc and 29 without (no-AVSc). The levels of oxidized (GSSG) and reduced (GSH) forms of glutathione were measured by a liquid chromatography-tandem mass spectrometry method and their ratio (GSH/GSSG) represent a recognized an oxidative stress status index. 2-AAPA, a glutathione reductase inhibitor, was used to increase GSSG levels. Human umbilical vein endothelial cells (HUVEC) were employed to create a model of protein glutathionylation. Valve endothelial cells (VEC) were isolated from pig and human aortic valves and were used to test and to validate this model.

Results:
Subjects with AVSc had a circulating GSH/GSSG ratio lower than no-AVSc (17.0±1.7 vs 24.4±2.6, respectively; p=0.02), indicating an increased systemic oxidative stress status in AVSc. Protein glutathionylation was increased more than 9-fold in human specimens from AVSc compared to no-AVSc subjects (p=0.03). In HUVECs, 2-AAPA significantly reduced the GSH/GSSG ratio (p=0.003) and induced an increment in protein glutathionylation of 1.91±0.17 fold compared to untreated cells (p=0.006). In pig VECs, 2-AAPA caused a 2-fold increment in reactive oxygen species production and an activation of the histone H2AX. In human VECs, protein glutathionylation caused an upregulation of smooth muscle actin (+0.7±0.16; p=0.004) and a downregulation of vascular endothelial cadherin (-0.4±0.02; p<0.0001) and, after 7 days, treated VECs were able to calcify at a greater extent (+2.83±0.4) than untreated cells (p<0.0001).

Conclusion:
These preliminary results show for the first time that protein glutathionylation could play a direct role in CAVD development via oxidative DNA damage. Restoring the proper balance of GSH and GSSG may be beneficial to reduce the endothelial damage occurring in patients affected by CAVD.
Abstract 468: Cross-species Comparison of Genetic Networks of Coronary Artery Disease
Authors: Kurt Zeyneb , Barrere-Cain Rio E , Sun Christine , Zhao Yuqi , Pan Calvin , Ruusalepp Arno , Franzén Oscar , Koplev Simon , Bjorkegren Johan L , Lusis Aldons J , Yang Xia
Published: March 2019

The genetic architecture of coronary artery disease (CAD), which represents the leading cause of death worldwide, has been investigated in both human populations and mouse models. However, whether the two species share similar genetic underpinnings has not been thoroughly evaluated. We hypothesize that a comprehensive data-driven integrative study leveraging multi-omics data resources will allow a tissue-specific, systems level assessment of the key similarities and differences in disease networks between mouse and man. To derive CAD genetic networks in mouse, we used genetic data from genome-wide association studies (GWAS) of atherosclerosis and functional genomics data of aorta and liver tissues from >100 strains profiled in an Atherosclerosis Hybrid Mouse Diversity Panel. To model human CAD genetic networks, we used human CAD GWAS from CARDIoGRAM+C4D, and functional genomics data from aorta and coronary arteries, and liver tissues from the Stockholm-Tartu Atherosclerosis Reverse Networks Engineering Task and Genotype-Tissue Expression biobanks. We identified coexpression network modules and biological pathways that were significantly associated with genetic signals in each species. This tissue-specific analysis revealed that ~75 and ~80% of the pathways and networks were shared between species for vascular and liver tissues, respectively. The shared processes between species for both tissues included lipid and lipoprotein metabolism, MAPK, TCA, and notch signaling pathways; for vascular tissues the shared terms included biological oxidations, interferon, and cytokine signaling; for liver tissue, fatty acid metabolism, TGF-beta signaling, leukocyte transendothelial migration were shared between species. Viral myocarditis, platelet-derived growth factor signaling, fibrinolysis, and IL6/7 pathways were identified as human-specific, while insulin and nerve growth factor signaling were found to be mouse-specific. This cross-species tissue-specific integrative analysis provides insights into the convergent and divergent causal mechanisms underlying CAD between species and highlights the conditions in which mouse may or may not serve as a surrogate model for human CAD in disease mechanistic and preclinical studies.
Abstract 469: APOL1 Genotypes and Risk of Heart Failure: an Additive Genetic Effect in the Elderly, but Not in the Young Adults in Patients with Chronic Kidney Disease
Authors: Liu Longjian , Chen Ming , Kim Yuna , Jia Hong , Long Yong , Zhong Yichen , Kelepouris Ellie , Eisen Howard J
Published: March 2019

Objective:
Apolipoprotein L1 (APOL1), the major protein component of HDL, is identified having an association with renal disease in African Americans (AA). We assessed the hypothesis that the genetic variations in APOL1 (G1 and G2 alleles) are associated with risk of heart failure (HF), and this effect is modified by age.

Methods:
Subjects aged 21-75, with an estimated GFR (eGFR) of 20 to 70 ml/min/1.73 m
2
at baseline recruitments (April 2003-Sept 2008), who were free of HF participating in the Chronic Renal Insufficiency Cohort (CRIC) Study and followed-up through March 2013 are analyzed (White: 1335, AA:1323). The associations of APOL1 risk genotype (assessed by either 1 of the G1 and G2, or 2 of their combinations, i.e., G1/G1, G1/G2 or G2/G2) with incident HF and mortality are examined using Cox’s regression models.

Results:
Within a mean 7-year (SD: 2.1) follow-up, AA had a significantly higher incidence of HF than White (2.62 vs. 1.67 per 100 person-year (PY), p=0.006) in those aged<65, and 3.94 vs. 2.71 per 100 PY (p=0.026) in those aged≥65. Cox’s models, with adjusted age, sex, eGFR and HDL, indicate that compared to White, AA aged<65 without or with 1 of either G1 or G2 had a significantly higher risk of HF [Hazard Ratio, HR: 2.15 (95%CI: 1.59-2.92, p<.0001), followed by those with either 2 of G1/G1, G1/G2 or G2/G2 alleles (1.47, 0.88-2.48, p=0.15)]. However, a significant additive genetic effect of the number of risk alleles (i.e., 0,1,2) is observed in AA aged ≥65. The corresponding HRs (95%CI) are 1.73 (1.17-2.57, p=0.007) in those without or with either 1 of the G1 or G2, and 2.17 (1.20-3.94, p=0.011) in those with either 2 of the G1 and G2 combination. A significant interaction effect of age (≥65 vs. <65) and the risk alleles (2 vs. 0/1) is observed (HR: 2.45, 95%CI: 1.14-5.28, p=0.02). Similar associations between APOL1 alleles and mortality from HF and all-cause are seen by the two age groups.

Conclusion:
African Americans with APOL1 risk alleles have significantly higher risk of HF and mortality than White. An additive genetic effect of the number of APOL1 risk alleles is shown among AA aged ≥65, but not in the younger adults. The modifying effect of age suggests that a possible 'different genetic-risk window' by ages warrants consideration in HF control and prevention.