bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 167: Vascular Tissue Engineering in Diabetes
Authors: Simionescu Agneta , Dhulekar Jhilmil , Hajdu Zoltan , Simionescu Dan
Published: July 2021

Tissue engineered blood vessels based on biological scaffolds seeded with autologous cells are currently being developed, but very little information exists regarding their fate in the complex diabetic environment. Diabetes is a major risk factor for vascular diseases as elevated levels of blood glucose and lipids interact irreversibly with long-lived proteins, such as collagen and elastin from the blood vessel wall, via oxidation and crosslinking, resulting in formation of advanced glycation end products (AGEs) and vascular stiffening. Vascular cells respond to diabetic environment by dysfunction and pathological remodeling, contributing to the onset and progression of vascular disease. These changes result in activation of inflammation, impaired healing, fibrosis, and calcification. Our hypothesis is that by reducing the level of AGEs in the cell milieu and stabilizing the matrix, vascular cells will positively contribute to the vascular wall remodeling. Therefore, acellular scaffolds prepared from renal arteries were treated with penta galoyl glucose (PGG), an antioxidant elastin-binding polyphenol, and seeded with adipose stem cells (ASCs) isolated from streptozotocin-induced diabetic rats. Constructs were analyzed for AGE formation, inflammation, and calcification after subnormal implantation as autologous cell seeded grafts, as well as by end-to-end anastomoses to the aorta. PGG-treated scaffolds showed good patency, undetectable dilatation and well-preserved elastin. ASCs seeded on scaffolds diminished macrophage infiltration, while simultaneously allowed M2 macrophage polarization. PGG-treatment and ASC seeding inhibited scaffold calcification and expression of osteogenic proteins. In conclusion, the antioxidative properties of PGG and the immunomodulatory properties of ASCs prevented vascular wall deterioration under the hostile diabetic milieu.
Abstract 171: Lysosomal Response Tocholesterol Loading is Altered in DBA/2 Mouse Foam Cells Which May Explain Impairedautolysosome Formation and Lipid Droplets Clearance
Authors: Robinet Peggy , Alzayed Alexander M , Smith Jonathan D
Published: July 2021

In a previous study, we identified autolysosome formation as the limiting step for turnover of cholesterol esters in lipid droplets of macrophage foam cells from the athero-sensitive DBA/2 strain compared to the athero-resistant AKR mouse strain. As autophagosome formation was similar in these two strains, we hypothesized that the lysosomal response to acetylated LDL (AcLDL) loading may be defective in DBA/2 vs. AKR foam cells. For all our studies, we cultured AKR and DBA/2 macrophages with or without AcLDL for 24h. AcLDL loaded DBA/2 vs. AKR cells exhibited a 40 to 50% decrease in lysosome number as measured by staining cells with Lysotracker or an anti-Lamp1 antibody, respectively. Lysosomal degradation capacity was assayed by cell incubation with Alexa647-DQ-ovalbumin, and we observed that AcLDL loading led to a 15% decrease in lysosomal capacity in DBA/2 foam cells (p<0.001) while it had no effect in AKR cells. Also, unloaded and loaded AKR cells had higher degradation capacity than DBA/2 cells (31% and 43%, respectively, p<0.001) which couldn’t be explained by a change in lysosomal pH. As the transcription factor TFEB is a key regulator for lysosome biogenesis and function, we analyzed TFEB protein expression by western blot. Upon loading, TFEB was increased in AKR (48%) but not DBA/2 cells leading to a 45% higher TFEB level in AKR vs. DBA/2 foam cells (p<0.05). AKR and DBA/2 macrophages were labeled with a TFEB antibody and the nucleus to cytoplasm fluorescence intensity ratio was assessed. AcLDL loading led to a 37% increase in TFEB nuclear localization in AKR foam cells vs. unloaded cells (p<0.001) with no effect in DBA/2 macrophages. These results were confirmed by western blot after cellular fractionation. In conclusion, we found that DBA/2 vs. AKR foam cells have altered TFEB processing that may explain decreased lysosome number and function and impaired autolysosome formation in DBA/2 cells.
Abstract 179: Computational Modelling Of Early Atherosclerotic Plaque Structure Reveals The Role Of The Rate Of Lipid Deposition On Plaque Structure And Growth Rate
Authors: Uddin Ishraq , Myerscough Mary R
Published: July 2021

Much current research on inflammation in atherosclerosis focuses on specific cellular and biochemical processes as isolated phenomena. There is scope, therefore, for an integrated approach that examines the interactions between the various mechanisms involved in early plaque growth. An in silico modelling approach is promising in this regard as multiple biological processes can easily be incorporated into the model and used to simulate early plaque growth under a variety of conditions. We present a computational model to simulate early plaque growth and test the hypothesis that higher rates of lipid deposition in the artery wall will cause accelerated plaque growth and influence the plaque’s morphology and composition.

We formulate a mathematical model based on the observed interactions between macrophages, pro-atherogenic extracellular lipid, lipid accumulated by macrophages, and apoptotic cells/cellular debris. This basic model incorporates several key processes including monocyte recruitment, phagocytosis, proliferation, apoptosis, lipid cytotoxicity, and cell migration. The model is used to computationally simulate how the early development of the plaque is influenced by its physiological environment, focusing in particular on the rate of lipid influx into the artery wall. Individual simulations yield a profile of how cells and lipids are spatially distributed over the plaque, and how this distribution and the total plaque size evolve in time.

The model qualitatively suggests that the rate of lipid deposition has a key influence on early plaque morphology and behaviour. Specifically, increasing the rate of lipid deposition (simulating higher fat diets) accelerates the rate of plaque growth. The ratio of apoptotic cells to active macrophages also increases with distance from the endothelium, with the ratio becoming more pronounced in older plaques. The computational model provides a novel methodology with which to capture the dynamics and structure of early atherosclerotic plaques, and provides a foundation on which to build models that can account for further aspects of the relevant biology, including plaque regression and necrotic core formation.
Abstract 180: Chronic Myelogenous Leukemia Cell Viability is Differentially Regulated by LXR Activation, HDL-associated Proteins, and LDL
Authors: Andersen Catherine J , Dupree Lydia , Doerr Adam , Cintron-Rivera Layra , Murray Kristina , Ragonesi Nicholas
Published: July 2021

LXR activation and HDL exert anti-cancer effects, albeit to varying degrees depending on cancer type and model. One of the mechanisms by which LXR and HDL impair cancer cell growth is through mediation of cellular cholesterol and phospholipid efflux, thereby limiting the availability of essential lipids for rapidly proliferating cells. Thus, the variability in LXR- and HDL-mediated anti-cancer effects may in part be due to differences in 1) HDL functionality and 2) the ability to induce expression of LXR target genes involved in lipid efflux and homeostasis due to adaptive shifts in cancer cell metabolism. Interestingly, serum amyloid A (SAA), a pro-inflammatory acute phase protein shown to be elevated in cancer, infection, and obesity, has recently been shown to differentially increase and decrease cholesterol efflux, despite its ability to displace apoA1 on HDL. Therefore, we investigated the effects of LXR activation, lipoproteins (HDL and LDL) and HDL-associated proteins (apoA1 and SAA) on the viability of chronic myelogenous leukemia cells - a cancer model previously unstudied in regards to cholesterol metabolism. Treatment of cells with the LXR agonist TO901317 (1μM, 5μM, and 10μM) significantly increased ABCA1 mRNA expression at 24hr and 72hr, in addition to dose-dependently decreasing HMG-CoA reductase and anti-apoptotic Bcl-xL mRNA expression. Changes in TO901317-induced gene expression corresponded to time- (24hr, 48hr, 72hr) and dose-dependent decreases in cell viability and average cell size. Interestingly, the addition of HDL to cell cultures with or without TO901317 treatment did not affect K562 cell viability, whereas LDL dose-dependently increased cell viability. Conversely, human-derived apoA1 (25 μg/mL) or HDL + apoA1 drastically reduced TO901317-treated K562 cell viability after 48hr and 72hr. K562 cell viability was modestly decreased by SAA (10 μg/mL) treatment, with greater decreases observed in cells treated with SAA + HDL. These findings suggest that LXR activation, HDL-associated proteins, and LDL differentially regulate chronic myelogenous leukemia cell viability.
Abstract 192: Intra-Individual Temporal Variation of Lipid and Apolipoprotein Levels in Patients With Type II Diabetes Mellitus and Normolipidemic Individuals
Authors: Parks Bryan A , Kuklenyik Susan , Schieltz David , Toth Christopher , Andrews Michael , Jones Jeffrey , Gardner Michael , Rees Jon , McWilliams Lisa , Barr John R
Published: July 2021

Hypertriglyceridemia is frequently observed with diabetes mellitus (DM) and is associated with increased cardiovascular disease (CVD) risk. In conditions of insulin deficiency or resistance, increased levels of free fatty acids are seen by the liver, which trigger increased lipogenesis and secretion of triglyceride (TG) rich lipoprotein (TRL) particles. However, a high number of specific ApoC-III on TRL particles suppress their return to the liver leading to a continuous circulation of highly atherogenic TRL remnants. To better understand the effect of intra-individual temporal variations on the differentiation of lipid and apolipoprotein profiles in DM vs. normal individuals, a small time course study over a 4 week period was performed on 3 normolipidemic, and 3 type II DM patients. Our laboratory has developed a method to separate lipoproteins based on hydrodynamic size coupled with mass spectrometry based analysis. Whole serum and lipoprotein size fractions were analyzed for 8 apolipoproteins (ApoA-I, ApoA-II, ApoA-IV, B-100, ApoC-I, ApoC-II, ApoC-III and E) and non-polar lipids (FC, CE, TG) while monitoring other proteins (ApoD, ApoM, CETP, LCAT, PLTP, PON1 and SAA4). In whole serum over the course of 4 weeks, ApoA-IV levels of DM vs. normal subjects differed the most, 3.2 (0.7) vs. 1.8 (0.4) μM, respectively, more significantly than intra-group or intra-individual variations. On the contrary, significantly lower whole serum levels were found for FC, CE, ApoM and PON1 (Prob<0.01). HDL-ApoC-III/LDL-ApoC-III was also lower in DM vs. normal subjects, 3.0 (1.9) vs. 7.2 (4.1). By metrics of molar ratio measured in 20-35 nm LDL fractions, the most significant difference was found between DM vs. normal subjects in ApoC-III/ApoB-100 (1.8 (0.6) vs. 0.9 (0.3)), C-II/ApoB-100 (0.8 (0.4) vs. 0.4 (0.2)) and ApoE/ApoC-III (0.11 (0.09) vs. 0.24 (0.15)). These LDL composition differences indicate lower lipase activity and inhibition of LDL uptake. However, no significant differences were observed in these molar ratios as function of LDL particle size. In conclusion, due to less significant temporal deviations, apolipoprotein molar ratios are a more useful and informative measure of CVD risk in DM subjects than LDL size distribution characteristics.
Abstract 200: Functional Mapping of the 8q24 Noncoding Region Reveals Enhancer Elements and Association Between GWAS SNPs and
TRIB1
Gene Expression
Authors: Berrido Andrea M , Smith Mikhaila A , Hand Nicholas J , Brown Christopher D , Rader Daniel J , Bauer Robert C
Published: July 2021

Single nucleotide polymorphisms (SNPs) in the human 8q24 locus have repeatedly been associated by genome-wide association studies (GWAS) with multiple human metabolic traits such as plasma lipids and coronary artery disease. These SNPs lie in a non-coding region ~30kb downstream of the gene Tribbles-1 (
TRIB1
). While a large body of in vivo evidence from
Trib1
gain- and loss-of-function mouse models strongly supports
TRIB1
as the gene of interest at this locus, there has been no demonstrated association between SNPs in the GWAS region and the expression of the neighboring
TRIB1
gene to date. To address this, we performed RNA-seq and genome-wide genotyping on 42 human cadaveric liver samples, and next performed allele-specific expression (ASE) analysis of the
TRIB1
locus in 23 samples that harbored heterozygous coding SNPs in the
TRIB1
gene, allowing for allelic discrimination. While subjects homozygous for the major allele of the lead GWAS SNP (rs2954029) had even allelic expression (
p
=0.29 by non-parametric t-test), samples heterozygous for the minor allele at rs2954029 exhibited imbalanced allelic expression (
p
<0.0005). This finding suggests that SNPs in the
TRIB1
locus do affect
TRIB1
gene expression; however, it remains unclear which SNP(s) is causal. To that end, we used ENCODE data from primary hepatocytes and HepG2 cells to identify 10 genomic regions of interest that contained SNPs with significant GWAS p-values and epigenetic markers consistent with enhancer activity. Two regions (R2, R9) exhibited strong enhancer activity when cloned in front of a minimal promoter driving a luciferase reporter (pGL4.23), increasing luciferase activity 2-5-fold as compared to empty vector (
p
<0.05). Introduction of the minor alleles at three separate candidate SNPs in R2 reduced enhancer activity. In summary, we show that common variation in the 8q24 GWAS locus does affect
TRIB1
gene expression, as measured by ASE in human livers. We also identified multiple enhancer elements that exhibit reduced activity when the minor alleles of GWAS SNPs are introduced into them. We are currently pursuing modification of the endogenous locus in HepG2 cells via CRISPR/Cas genome editing to further elucidate the roles of both these enhancer elements and the SNPs contained in them.
Abstract 210: Milk Fat Globule Epidermal Growth Factor VIII Mediates Vascular Remodeling by Increasing Inflammation and Smooth Muscle Activation
Authors: Lee Ting-Hein , Nagata Shigekazu , Atabai Kamran , Chiang Hou-Yu
Published: July 2021

Vascular remodeling, defined as a change in the geometry of the vessel wall, occurs in the pathological process of vascular diseases, like atherosclerosis, hypertension and restenosis. The resulting neointimal formation is a part of a reparative response including thrombosis, inflammatory cell infiltration, vascular smooth muscle cell (VSMC) proliferation and migration, which lead to the stenosis of blood vessels and the restricted blood flow. Milk fat globule epidermal growth factor VIII (Mfge8), a secreted glycoprotein, is well-characterized for its capacity of assisting the clearance of apoptotic cells in vascular system. Recently, Mfge8 has been identified as a pivot relay between pro-inflammatory signals and activated VSMCs, contributing to intima-media thickening of the vessel wall by promoting VSMC proliferation and migration in aged arteries. We have noted intense Mfge8 expression in the endothelial cells and VSMCs of the carotid artery following ligation injury in mice, suggesting that Mfge8 may regulate the two characteristics of vascular remodeling, inflammatory cell infiltration and VSMC activation, in response to low blood flow. To elucidate the functions of Mfge8 in a flow-induced model of vascular remodeling, a complete carotid ligation was conducted in wild-type (WT) or Mfge8 knockout (KO) mice. Morphometric analysis demonstrated that genetic deletion of Mfge8 in mice reduces carotid intima and media thickening compared to WT mice. Deficiency of Mfge8 prevented VSMC phenotypic modulation, as evidenced by the decreased expression of smooth muscle myosin heavy chain and attenuated cell proliferation in tunica media after ligation injury. VSMCs transfected with SiRNA against Mfge8 migrated slower than in controls as early as 0.5 days post-platelet-derived growth factor (PDGF) stimulation. Further, Mfge8-null mice showed a dramatic decrease in leukocyte infiltration into the vessel wall. Collectively, in a flow-induced model of vascular remodeling, Mfge8 plays a crucial role in VSMC migration and proliferation, as well as inflammatory cell accumulation, thereby regulating neointimal formation.
Abstract 213: Deletion of Epha2 Reduces Fibroproliferative Remodeling in Atherosclerosis and Vascular Smooth Muscle
Authors: Finney Alexandra C , Green Jonette M , Rana Mohammad A , Funk Steven D , Traylor James G , Orr A. Wayne
Published: July 2021

Advanced atherosclerosis involves the recruitment of vascular smooth muscle cells, leading to smooth muscle-mediated fibroproliferative remodeling and plaque stiffening. We previously demonstrated the neuronal guidance receptor EphA2 shows enhanced expression in murine and human atherosclerosis, and EphA2 deletion in atherosclerosis-prone ApoE knockout mice attenuates lesion formation and endothelial cell activation in models of atherosclerosis. We now demonstrate deletion of EphA2 attenuates plaque progression to advanced stages associated with diminished smooth muscle content. While contractile smooth muscle cells show minimal EphA2 expression
in vitro
and
in vivo
, transition to the synthetic phenotype results in enhanced EphA2 expression and luciferase reporter activity
in vitro
and in atherosclerotic lesions. Furthermore, forced expression of EphA2 in quiescent smooth muscle cells is sufficient to suppress contractile smooth muscle markers, suggesting a role for EphA2 in smooth muscle phenotypic switch. Depletion of EphA2 results in significant reductions in proliferation within atherosclerotic plaques and in smooth muscle cells with associated reductions in serum-induced ERK and Akt signaling. Conversely, overexpression of EphA2 enhances smooth muscle proliferation
in vitro
, suggesting EphA2 is sufficient to drive proliferation. EphA2-deficient mice show reductions in plaque fibrosis and matrix remodeling. Consistent with this, depletion of EphA2 in vascular smooth muscle cells exhibit a significant reduction in matrix deposition. Together these data suggest a role for EphA2 in smooth muscle-mediated fibroproliferative remodeling, representing the first link between EphA2 signaling and smooth muscle function in atherosclerosis.
Abstract 215: Rac-Signaling as a Critical Determinant of IL-1beta-Dependent Atherosclerotic Calcification
Authors: Healy Abigail L , Ceneri Nicolle , Mantsounga Chris , Morrison Alan R
Published: July 2021

Coronary artery disease caused by atherosclerosis is the leading cause of morbidity and mortality in the world. Calcification of atherosclerotic plaque has predictive value in terms of cardiovascular event risk and mortality. Plaque calcium composition appears critical to determining cardiovascular risk; microcalcification has been associated with more vulnerable plaque phenotypes, while somewhat paradoxically, densely calcified plaque is associated with more stable disease. Inflammation can influence calcification of plaque, but immune modulators of plaque calcification are minimally defined. Rac1 and Rac2 are small GTPases that influence cytokine expression in plaque macrophages. We have defined a Rac-based signaling mechanism that regulates macrophage IL-1β expression. Using an atherosclerotic-prone mouse model, we identified that progressive calcification depends on altered Rac expression and activation (GTP-binding) through consequent effects on macrophage IL-1β production and IL-1R signaling. IL-1β production and its downstream signaling were critical determinants of atherosclerotic plaque calcification. In short, Rac2 expression determined the degree of Rac1 GTP-binding, acting as a brake on Rac1-dependent IL-1β production, whereas macrophage IL-1β production and atherosclerotic calcification depended on myeloid Rac1. Therapeutic inhibition of macrophage Rac-mediated IL-1β expression has the potential to be a treatment strategy for progressive, inflammatory atherosclerotic calcification.
Abstract 216: Hdac2 Modulates Atherosclerosis
Authors: Pandey Deepesh , Hori Daijiro , Nomura Yohei , Bhatta Anil , Berkowitz Dan , Romer Lew
Published: July 2021

Objective:
We recently described a novel regulatory role for histone deacetylase 2 (HDAC2) in vascular endothelial cell protection against the known endothelial injury stimulus Oxidize Low Density Lipoprotein (OxLDL). The goal of current study is to determine the effects of endothelial-specific HDAC2 overexpression on endothelial dysfunction and atherogenesis
in vivo
.

Approach and Results:
To study the effect of endothelial HDAC2 overexpression on vascular function and atherogenesis, we generated and characterized an endothelial specific human HDAC2 overexpressing transgenic mouse (HDAC2-Tg) under the control of the tie2 promoter. The founder HDAC2-overexpressing mice appear to have a normal phenotype- normal weight, mean arterial blood pressure (MAP) and pulse wave velocity (PWV). Isolated intact aortas from endothelial-specific overexpressing HDAC2 transgenic mice (HDAC2-Tg) exhibited a healthier vascular function profile with higher nitric oxide levels and less OxLDL-mediated vascular dysfunction. Further, we induced atherogenesis in C57BL6 and HDAC2-Tg mice by injecting adeno-associated viruses (AAV) encoding PCSK9 (D377Y) under the control of a liver-specific promoter by tail vein injection followed by high fat diet regimen for 12 weeks and measured endothelium-dependent vasorelaxation in isolated blood vessels, pulse wave velocity, an index of vascular stiffness and mean arterial blood pressure. HDAC2-Tg exhibited protected endothelial dependent vascular relaxation and no significant changes in MAP and PWV as compared to control mice.

Conclusion:
HDAC2 is a critical regulator of endothelial function. Overexpression or activation of HDAC2 represents a novel therapy for endothelial dysfunction and atherosclerosis.
Abstract 221: B-1b Cells Produce IgM to Malondialdehyde-Modified Low Density Lipoprotein in Perivascular Adipose Tissue in Response to Immunization And Attenuate Diet Induced Atherosclerosis
Authors: Srikakulapu Prasad , Upadhye Aditi , Rosenfeld Sam M , Kouhestani Kaivon , Marshall Melissa , Prohaska Thomas A , Witztum Joseph L , McNamara Coleen A
Published: July 2021

Background:
B-1b cells are capable of long-lasting IgM memory and secrete more IgM, particularly malondialdehyde-modified low density lipoprotein (MDA-LDL) specific IgM than B-1a cells after transfer into hyperlipidemic Rag1
-/-
mice. Id3 is a basic helix-loop-helix protein and dominant negative inhibitor of E proteins. B cell specific Id3 deficiency (Id3
BKO
) increases B-1b cell numbers systemically and provides atheroprotection. Adipose tissue is a source of B-1b-derived IgM and regulates inflammatory cytokine production from M1 macrophages locally. Perivascular adipose tissue (PVAT) has been implicated in regulation of atherosclerosis. However, the effect of B cell specific Id3 deficiency on B-1 cell responses in PVAT is not yet known. Also, whether these B-1b cells respond to MDA-LDL immunization is unknown.

Hypothesis:
B-1b cells are present in PVAT and produce MDA-LDL specific IgM in PVAT, and immunization with MDA-LDL can enhance B-1b-mediated atheroprotection.

Methods and Results:
Flow Cytometry and Enzyme-Linked ImmunoSpot (ELISPOT) analysis of PVAT of normal chow diet fed young mice demonstrated that ApoE.Id3
BKO
mice have significantly higher numbers of B-1b cells and IgM secreting cells but not B-2 and B-1a cell numbers in PVAT compared to ApoE.Id3
WT
mice. ELISPOT demonstrated that the % of MDA-LDL specific IgM of total IgM secreting cells was significantly greater in PVAT, but not in spleen or bone marrow, of ApoE.Id3
BKO
mice compared to ApoE.Id3
WT
mice, suggesting that modified lipids such as MDA-LDL in plaques and PVAT may stimulate local B-1b cells to secrete MDA-LDL specific IgM. Adoptive transfer of B-1b cells into ApoE.Rag1
-/-
mice followed by MDA-LDL+PPS3 (pneumococcal polysaccharide) immunization, increased plasma IgM to MDA-LDL after 2 weeks and significantly attenuated atherosclerosis after 16 weeks of Western diet compared to PBS injected mice and B-1b cells transferred mice with PBS immunization.

Conclusion:
B-1b cells produce IgM to MDA-LDL in PVAT. MDA-LDL immunization increased IgM to MDA-LDL after two weeks and attenuated diet-induced atherosclerosis. Taken together, results suggest that B-1b cells may regulate atherosclerosis through both local and systemic IgM production.
Abstract 222: Utility of Non-Contrast, Non-EKG Gated Multi-Detector Computed Tomography to Rule Out Obstructive Coronary Artery Disease in Heart Transplant Recipients
Authors: Thakkar Akanksha , Gonzalez-Bonilla Hilda Mariana , Senapati Alpana , Gupta Nakul , Alvarez Paulino , Chang Su Min
Published: July 2021

Background:
Annual or biannual coronary angiography is recommended after heart transplantation. This is associated with radiation and contrast exposure in addition to being invasive. Absence of coronary calcium on CT in non-transplanted patients has a high negative predictive value (NPV) to rule out obstructive CAD.

Objective:
To determine whether the absence of coronary calcium on non-contrast, non-gated MDCT has a high NPV to rule out obstructive CAD in heart transplant recipients.

Methods:
We included patients with a non-contrast, non-gated chest CT within 1 year of undergoing a coronary angiogram from 2006 to 2016. Obstructive CAD was defined as stenosis >50%. Coronary artery calcium scores (CACS) were calculated by the Agatston method using the syngo.via platform (Siemens Healthcare GmbH, Germany). For analysis, CACS were categorized based on a) presence or absence of coronary calcium (i.e., CACS=0 or CACS>0) or b) severity of calcification (i.e., CACS=0, CACS 0-100 and CACS>100).

Results:
We reviewed 76 consecutive angiograms and corresponding CTs. Prevalence of obstructive CAD was 9.2% (n=7). Prevalence of CACS=0 was 89.5% (n=68) of which 5 had obstructive CAD presenting a NPV of 93%. 8 patients had CACS>0 of which 2 had obstructive CAD on angiogram (positive predictive value=25%). CACS was found to correlate with severity of CAD by angiography(p<0.05). Of 7 angiograms with obstructive CAD, stenosis >50% was found in 10 vessels - 5 LAD, 1 D1, 1 LCx, 2 OM branches and 1 RCA. Calculating CACS enabled detection of 1/5 LAD lesions; lesion in D1 was detected as an LAD lesion; lesions in the OM branches and RCA were not detected.

Conclusion:
Absence of coronary calcium on MDCT has a high NPV in ruling out obstructive CAD in heart transplant recipients. However, only 2/10 obstructive lesions on angiogram had calcification plaques detected on corresponding vessels on CT. The clinical significance of this is yet to be determined.
Abstract 244: Distinct Regulation of Fat and Dachsous Cadherins in Response to Arterial Injury
Authors: Riascos-Bernal Dario F , Cao Longyue L , Chinnasamy Prameladevi , Hou Rong , Sibinga Nicholas E
Published: July 2021

Molecular mechanisms that control the activities of vascular smooth muscle cells (VSMCs) in the diseased or injured arterial wall remain incompletely understood. The atypical cadherin FAT1 is prominently expressed in VSMCs after vascular injury. In recent work, we found that processing of FAT1, a type I transmembrane protein, releases its intracellular domain, the FAT1ICD; in turn, FAT1ICD fragments accumulate in mitochondria and interact with electron transport complexes I and II to restrict VSMC respiration and control cell growth and neointimal formation. We hypothesized that FAT1 processing (and therefore these VSMC activities) is controlled by extracellular ligands that may interact physically via FAT1’s extended cadherin repeat or transmembrane domains. FAT4 and Dachsous 1 (DCHS1) are leading candidates for such interactions, but these proteins have not been studied in VSMCs or vascular injury response. Accordingly, we analyzed expression profiles of FAT4 and Dachsous 1 (DCHS1) during the vascular response to injury, using a rat carotid artery balloon injury model. Interestingly, FAT4 transcripts increased transiently after injury, peaking at day 7 (6.99±0.21-fold over baseline, P<.001), in a pattern reminiscent of FAT1. In contrast, DCHS1 levels decreased by day 3 after injury (0.49±0.02-fold of baseline, P<.001), remained low through day 14, and recovered by day 30. Physical interactions of FAT4 either with DCHS1 or with FAT1 have both been reported previously; therefore, decreased DCHS1 coincident with increased FAT4 suggest an increase in FAT4 availability and/or interaction with FAT1 during injury response. How this increase in FAT4 affects FAT1 function and VSMC metabolism and growth is the subject of ongoing investigation. FAT1 induction and processing after vascular injury represent an important novel molecular mechanism by which VSMC metabolism and growth are controlled after vascular injury. Increased availability of FAT4 cadherin due to FAT4 induction together with DCHS1 downregulation after vascular injury provides a likely upstream regulatory mechanism to govern FAT1 activities.
Abstract 247: The Association of the Serotonin Transporter Polymorphism LL-Genotype with Non-Syndromic Mitral Valve Prolapse Requiring Surgery at a Younger Age
Authors: Driesbaugh Kathryn H , Grau Juan B , Lai Eric K , Branchetti Emanuela , Rioux Nancy , Alkhaleel Farrah , Frick Nathan , Levy Robert J , Ferrari Giovanni
Published: July 2021

Background:
Non-syndromic mitral valve prolapse (MVP) is a highly prevalent heart valve disease that is treated surgically when indicated by clinical deterioration. Serotonin (5HT)-related valvulopathies have been reported; however, 5HT has not been shown to be directly associated with MVP. A polymorphism (5-HTTLPR), with a 44-base micro-insertion/deletion in the promoter of the 5HT transporter (SERT) gene, is present in all human populations studied. 5-HTTLPR, with designated long (L) or short (S) alleles, has been reported to have an impact on SERT expression in non-cardiac valve studies. We hypothesized that MVP surgical patients may have an altered distribution of 5-HTTLPR, resulting in SERT expression levels associated with an increased risk of disease progression.

Methods:
201 MVP surgical patients were recruited under an IRB approved protocol; DNA extraction followed by genomic fragment analysis was performed to determine allelic frequencies. qRT-PCR of SERT and related genes was carried out on RNA extracted from available MVP tissue (N=64). A RT2 Profiler microarray study for 84 genes associated with human dopamine and 5HT signaling was also performed (N=44).

Results:
MVP surgical patients had an overall frequency of LL-32%, LS-50%, and SS-18%. The frequency of 5-HTTLPR-LL was significantly increased in males under 55 years of age (N=19/41, 47.6%) (
p=0.04
) compared to the rest of the population. Interestingly, the LL patients had the lowest SERT expression levels per qRT-PCR compared to LS and SS. A similar expression pattern was also noted for another 5HT transporter, vesicular monamine transporter-2 (VMAT2), with the lowest expression in the LL specimens. Expression of 5HT receptors 2A and 2B were comparable between genotypes in the MVP samples analyzed. Similarly, microarray studies demonstrated tight regulation of 5HT-associated genes, but with LS/SS patients expressing >2-fold more SERT than LL.

Conclusion:
5-HTTLPR-LL is associated with MVP surgery at a younger age in men, with relatively lower expression of both SERT and VMAT2, suggesting a reduced capacity for transporter processing of 5HT. The 5-HTTLPR-LL genotype may constitute a novel risk factor useful for identifying MVP patients more likely to have rapid disease progression.
Abstract 248: Comparative Transcriptomic Analysis of Smooth Muscle Cells and Endothelial Cells Identifies Distinct Signaling Networks Following Inflammatory Challenges
Authors: Wang Bowen , Pan Xiaokang , Zhang Mengxue , Shi Xudong , Guo Lian-Wang , Kent K C
Published: July 2021

Background:
Drug-eluting devices have shown promising outcomes in patients receiving endovascular procedures, particularly in managing post-intervention restenosis that often leads to failure of the reconstructions. However, emerging evidences have revealed increased thrombogenic risk associated with the use of drug-eluting stents and balloons. It has been widely acknowledged that the major culprit responsible for the observed thrombosis events is the non-selective damage of the anti-restenotic compounds on vascular endothelial cells (ECs), which, under physiological conditions, serve as the protective barrier for maintaining vascular homeostasis. Given the clinical relevance, there is an urgent need for a therapeutic agent that could confer selective management of smooth muscle cells (SMCs) without damaging ECs. However, by far the differential mechanisms in the two cell types underlying their distinct cellular behaviors toward pathogenic stimuli are poorly understood. Thus, a systematic and comparative analysis of their cellular dynamics at the transcriptomic level is needed.

Methods and Results:
Human primary aortic SMCs and ECs were starved and then stimulated with 2 pro-inflammatory cytokines (TNF-α and IL-1β), which are well-established inducers of SMCs’ proliferative and migratory phenotypes while simultaneously damaging ECs. Samples were then subject to RNA sequencing. We developed a customized algorithm to evaluate the differential responses and transcriptomic network dynamics in the two cell types, and successfully identified multiple gene modules that contain functionally related genes that possibly are involved in the distinct regulations of SMCs versus ECs post inflammatory challenges.

Conclusions:
Our study provides the first comprehensive analysis of the differential transcriptomic dynamics between SMCs and ECs following inflammatory challenges, which are prominent contributors to the pathogenesis of both restenosis and thrombosis following vascular injury. Our data identify several module groups of genes that could serve as potential targets to achieve differential modulation of the pathophysiologies of SMCs versus ECs. Further studies are warranted to validate the contributions of these genes.
Abstract 251: Loss of Endothelial E Prostanoid Receptor 4 Exacerbates Wire-Injury Induced Neointimal Formation
Authors: Hao Huifeng , Hu Sheng , Chen Hong , Wang Miao
Published: July 2021

Prostanoids, which are synthesized from the cyclooxygenase cascade and inhibited by nonsteroidal anti-inflammatory drugs, participate in vascular remodeling. Deletion of microsomal prostaglandin (PG) E synthase-1 increases prostacyclin and attenuates injury-induced neointimal formation in mice. The role of E prostanoid receptor 4 (EP4) in vascular response to injury is unknown. Using an inducible Cre-LoxP-based approach, we generated the endothelial-restricted EP4 knockout mice (cKO) and their littermate controls (Ctl). After tamoxifen treatment, one side femoral arteries of the mice were subjected to wire-injury. Twenty-eight days later, the femoral arteries were harvested and the neointima formation were evaluated. Deletion of endothelial EP4 strikingly exacerbated intima area and the ratio of intima to media area, without affecting media area. Treatment of wild type mice with AE1-329, a selective EP4 agonist, significantly reduced neointimal hyperplasia after wire-injury. Mechanistically, activation of endothelial EP4 signaling with AE1-329 promoted endothelial cell proliferation. Notably, the adhesion of leukocytes to endothelial cells was significantly inhibited by AE1-329 treatment. Collectively, endothelial EP4 may protect against vascular remodeling via enhancing endothelial repair and reducing leukocytes adhesion to the endothelium.
Abstract 253: The Direct Renin Inhibitor Aliskiren Prevents Endothelial Dysfunction in Spontaneously Hypertensive Rat Aortas
Authors: Tse Gary , Tian Xiaoyu , Huang Yu , Wong Jack
Published: July 2021

Introduction:
Hypertension is a significant medical problem. Aliskiren is a novel anti-hypertensive agent that inhibits renin directly. We tested the hypothesis that aliskiren prevents endothelial dysfunction in spontaneously hypertensive rats (SHRs).

Methods:
SHRs and age-matched Wistar Kyoto (WKY) rats were randomly divided and subjected to oral administration of aliskiren (10 mg/kg/day) or vehicle for 8 weeks. Blood pressure was monitored bi-weekly by a tail-cuff method. After an initial equilibration period, each ring was contracted by elevated KCl (60 mmol/L) to ensure a level of contractility suitable for subsequent experimentation. To examine endothelium-dependent relaxations in phenylephrine-contracted rings, two consecutive concentration-response curves to acetylcholine (ACh; 3 nmol/L -30 μmol/L) were constructed. In some phenylephrine-contracted rings without intact endothelium, sodium nitroprusside (SNP; 3 nmol/L – 10μmol/L) were applied cumulatively to the bathing solution to test the sensitivity of vascular smooth muscle cells (VSMCs) to NO.

Results:
Aliskiren reduced systolic blood pressure (SBP) to 155.6 ± 1.87 mmHg as compared to 165.3 ± 3.2 mmHg in vehicle-treated SHRs (n = 7), while chronic aliskiren administration did not affect SBP in WKY rats. SHR aortas with endothelium relaxed less in response to different concentrations of ACh than age-matched WKY rat aortas and ACh failed to cause relaxations of aortic rings without endothelium. By contrast, endothelium-independent relaxations caused by sodium nitroprusside (SNP) were comparable in aortas from SHR and WKY rats. Chronic aliskiren administration improved endothelium-dependent dilatations of aortas from SHRs compared to vehicle-treated SHRs, but this was unaffected in WKY rats. Endothelium-independent relaxations to SNP were not significantly different in aortas from the four treatment groups.

Conclusions:
Aliksiren reduces blood pressure and improves endothelium-dependent dilatations of SHRs.

Figure 1.
Effects of 8-week treatment of aliskiren on acetylcholine (ACh)-induced endothelium-dependent dilatations in SHR
(A)
and WKY
(B)
aortas. Results are mean ± SEM of 7 experiments. *p<0.05.
Abstract 256: Differential Biomechanical Regulation of Vein Graft Endothelial CD39 by Pulsatile Radial Forces
Authors: Kanthi Yogendra , Yadav Vinita , Vear Kinsey , Jacobs Benjamin N , Hao Yibai , Liao Hui , Pinsky David J
Published: July 2021

Saphenous veins are frequently used bypass grafts in coronary & peripheral artery bypass surgery, but are more prone to failure than arterial grafts due to adverse remodeling. The endothelium senses radial and linear biomechanical forces exerted by blood flow. Thin-walled venous blood flow
in situ
is non-pulsatile until transposed as an arterial graft. The impact of cyclic stretch from pulsatile distention on the vein wall is not well elucidated. CD39 is a potent ecto-enzyme at the blood:vessel interface regulating vascular thrombo-inflammation by dissipating extracellular ATP & ADP. We recently identified that vascular CD39 expression can be induced by laminar shear stress. However, the response of CD39 to cyclic stretch during vein graft arterialization remains unknown. We
hypothesized that the vasculo-protective enzyme CD39 is induced by the venous endothelium in response with pulsatile stretch.

Methods:
Primary human saphenous vein endothelial cells (HSVEC) were exposed to cyclic stretch
in vitro
, mimicking arterial & vein graft patterns. CD39 transcript & protein expression were quantified. Ecto-ATPase and -ADPase activity of CD39 on HSVEC were quantified with a malachite green assay. CD39 haploinsufficient (

Cd39
+/-

) & WT mice underwent carotid bypass surgery with a vein graft, & subsequent neointimal hyperplasia was compared.

Results:
High (16%, 1 Hz) and low (5%, 1 Hz) levels of cyclic stretch of HSVEC elicited distinct patterns of CD39 expression.
Cd39
mRNA remained unchanged after 48h of low or high stretch (n=6,each). Low-level cyclic stretch did not alter CD39 protein expression (n=6,each). In stark contrast, 16% cyclic stretch induced increases in CD39 protein expression after 24h (60%) and 48h (130%) compared with static controls (n=6 each,
P
<0.05). CD39-dependent ATP & ADP hydrolysis increased by 60% & 100% in HSVEC following high-level cyclic stretch (n=4, p<0.005). Preliminary murine vein graft model studies suggest that CD39-deficiency exacerbates vein graft neointimal hyperplasia compared to WT controls.

Conclusions:
We have identified CD39 as a novel, stretch-responsive endothelial ecto-enzyme in human saphenous veins, which may represent a protective response to venous distention from arterial stretch patterns.
Abstract 262: Drebrin Regulates Aortic Remodeling in Angiotensin II-Induced Hypertension
Authors: Zhang Lisheng , Wu Jiao-Hui , Huang Tai-Qin , Nepliouev Igor , Brian Leigh , Zhang Zhushan , Rudemiller Nathan P , Crowley Steven D , Freedman Neil J , Stiber Jonathan A
Published: July 2021

The actin-binding protein, Drebrin, is upregulated in response to arterial injury and reduces smooth muscle cell migration/proliferation through its interaction with the actin cytoskeleton. Because hypertensive aortic remodeling involves smooth muscle cell (SMC) activation and synthesis of extracellular matrix, we tested the hypothesis that Drebrin inhibits this process. To determine the effect of Drebrin deficiency in SMCs on hypertensive aortic remodeling, we induced hypertension by implanting osmotic mini-pumps to infuse angiotensin II (Ang II, 1000 ng/kg/min) or vehicle (0.9% NaCl) continuously for 28 days in SM22-α Cre
+/-
;
Dbn
flox/flox
mice (SMC-
Dbn
-/-
mice) and controls. Blood pressure (BP) responses to Ang II treatment were assessed by telemetry. After completion of Ang II infusion, the degree of aortic remodeling was assessed by computerized tomography of histologic cross-sections of the proximal ascending aorta. Despite observing no difference in the extent of Ang II-induced hypertension in SMC-
Dbn
-/-
mice compared with controls, SMC-
Dbn
-/-
mice exhibited a significant increase in medial hypertrophy and outward remodeling. Wall thickness/body weight was increased by 61 ± 2% (p<0.01) in SMC-
Dbn
-/-
mice compared with controls, and lumen area/body weight was increased by 102 ± 9% (p<0.01). Cellular proliferation and matrix deposition were increased in the proximal aortas of Ang II-treated SMC-
Dbn
-/-
mice compared with controls as evidenced by increased immunoreactivity for PCNA, p-ERK, and Collagen I. SMC loss of Drebrin also resulted in increased Ang II-induced pro-inflammatory signaling as evidenced by increased expression of VCAM-1, p-P65 and MMP-9 and increased CD68 positive cellular proliferation in response to chronic Ang II infusion. We conclude that SMC loss of Drebrin augments adverse aortic remodeling in angiotensin II-induced hypertension.
Abstract 266: Extracellular Hemoglobin From Stored Rbcs Binds Ago2 And Delivers Functional Microrna to Endothelial Cells
Authors: Mohandas Appesh N , Thomas Sheena A , Rooney Kimberly A , Mitchell Adam J , Roback John D , Searles Charles D
Published: July 2021

Red blood cell (RBC) transfusion is among the most common clinical therapies. Prior to transfusion, packed RBCs (PRBCs) may be stored for up to 42 days. During storage, RBCs undergo many changes, including hemolysis and the release of extracellular hemoglobin (Hb). Prior work has shown that endothelial cells exposed to free hemoglobin demonstrate upregulation of inflammatory and anti-inflammatory pathways including elevated ICAM-1, VCAM-1 and E selectin, as well as elevated Hemeoxygenase-1 and bilirubin levels. Our work tests the hypothesis that extracellular Hb from stored RBCs can transfer functional microRNA to endothelial cells.

Stored RBCs (age 30-42 days) were obtained from the Emory University Hospital Blood Bank. Extracellular Hb was obtained from lysed RBCs. Binding of Argonaute 2 (Ago2) to Hb was assessed by immunoprecipitation (IP) of Hb-alpha followed by Western blot for Ago2. In addition, microRNA was extracted from the Hb immunoprecipitate. Levels of miR-451, -16, -584, and -92 were assessed by RT-qPCR analysis. Lastly, human aortic endothelial cells (HAECs) were treated with Hb from RBC lysates. After treatment, Qiazol was used to extract RNA from washed cells, and levels of miR-451 and target genes were measured by RT-qPCR and Western analysis.

We found that Ago2, a crucial component of the RISC complex that executes the regulatory function of microRNA, co-immunopreciptated with Hb from RBC lysates. We also observed that miR-451, a microRNA known to be abundant in RBCs, was highly abundant in Hb immunoprecipates. Furthermore, miR-16, -584 and -92 could also be amplified from Hb immunoprecipitates. Endothelial cells treated with extracellular Hb from RBC lysates for 36 hours had a dramatic increase in levels of miR-451 and a significant decrease in mRNA abundance of genes known to be targets of miR-451, including ATF2 (p-value=0.03), CAV1 (p-value=0.02), and MIF (p-value=0.005). HAECs treated with extracellular Hb for 72 hours had decreased protein levels of ATF2 (p-value=0.01) and CAV1 (p-value=0.003). In conclusion, previous work has shown that extracellular Hb can have direct effects on endothelial cells; our data show, for the first time, that extracellular Hb also serves as a vehicle for the cell-to-cell delivery of microRNA.
Abstract 301: Association of Short and Long Sleep Duration With Carotid Intima-Media Thickness, the Baptist Employee Healthy Heart Study (Behhs)
Authors: Aziz Muhammad , Kandimalla Yugandhar , Machavarapu Archana , Younus Adnan , Malik Rehan , Banala Mounica , Saxena Anshul , Humayun Choudhry , Pena Victor M , Jaber Rana , Veledar Emir , Nasir Khurram
Published: July 2021

Objective:
Carotid intima media thickness (CIMT) is well-known marker of cerebrovascular & CVD outcomes. Recent literature has discussed association of sleep duration with stroke &CVD, but still limited evidence exists regarding the true relationship of sleep duration with CIMT. The aim of this study is to determine association of short& long sleep duration with CIMT.

Method:
Baptist Health South Florida, a not for profit organization, conducted a randomized, non-blinded controlled trial in 2014.This study examined effect of web based interventions on reducing CVD risk in employees. The inclusion criteria were physician diagnosed T2DM and/or Metabolic Syndrome. We used cross sectional data for analysis. Per CDC.gov guidelines, we categorized self-reported sleep duration (hrs) as short (<7), reference (≥7-<9) and long sleep (≥9). CIMT was measured via carotid US screening device by Panasonic CardioHealth Station.

Result:
Study population (n=183; 74% female, 49% Hispanic) with mean age 51±10 years. Mean CIMT(mm) in females [0.879±0.15] and males [0.911±0.19] was not different (p>0.05). Atherosclerotic plaque was defined as any obvious focal luminal encroachment > 1.2 mm. In multivariate logistic regression model, per hour increase in sleep duration was associated with twice the odds of increase in CIMT >1.2mm [OR 2.15;95% CI (1.15-4.02)]. However, once we compared the reference sleep with short and long sleep duration categories, we determined, as compared to 7-9 hrs (ref) of sleep, the odds of CIMT >1.2 in those sleeping <7 hrs [OR 1.23; 95% CI (0.27-5.53)] and those sleeping ≥9 hrs [OR 2.91; 95% CI (0.33-25.42)] were not significant in adjusted model.

Conclusion:
Although we observed that per hour increase in sleep was related to increase in CIMT >1.2mm (risk of plaque), but we did not find any significant increased risk of plaque in either short or long sleep duration. Longitudinal studies with larger sample size are needed to clarify this association.