bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 361: Plant-Based Diet and Lifestyle Changes Improve Lipids Among Angina Patients
Authors: Ramirez Francisco E , Nedley Neil , Sanchez Albert
Published: July 2021

Background:
Angina is an important predictor of heart disease. Lipids can be an important etiological factor of atherosclerosis and angina. We examine the effect that an 18 day medical residential program has on the lipids of such patients.

Methods:
Participants completed an 18 day program in Weimar, California near Sacramento. Each patient was evaluated by a board certified physician who monitored them during the whole program. Blood samples were taken before and at the end of the program. Results are reported in mg/dl. Among the modalities used in all participants included whole foods plant-based diet, aerobic exercise, proper water intake and hydrotherapy, enough sunlight, avoidance of any addictive substances (tobacco, caffeine, alcohol, etc), breathing exercises, enough rest and psychological, relational and spiritual therapies. The meals were buffet style, 3 times a day with no snacks. Various cooking classes and daily educational lectures were given to empower patients to continue with the changes at home.

Results:
From
n
=2080 patients,
n
=133 had an angina diagnosis. From the 133 and 116 had complete lab results, 50% percent were males. Results are reported as (mean, SD, median, mode, min, max). Baseline total cholesterol (218, 41, 213, 189, 96, 316), end values (189, 33, 185, 163, 108, 283) t-test t(115)=11.4, with significant change
p
<.001, mean difference 28.3. Baseline LDL (137, 37, 128, 117, 53, 227), end values (115, 29, 112, 123, 55, 204), t-test t(115)=10.2, with significant change
p
<.001 mean difference 21.6. Baseline HDL (43, 15, 40, 31, 18, 90) end values (42, 12, 39, 33, 21, 76), t-test t(115)=1.2 change was not significant,
p
=.22, mean difference .92. Some patients reported decrease in angina symptoms.

Conclusions:
The program effectively decreases lipids towards a normal range. HDL decreased a little but the change was not significant. Long term follow up should be done on participants of this program to see the long term effect.
Abstract 364: An NPC1 and NPC2 Independent Pathway Mediates Trafficking of Cholesterol From Lysosomes to the Extracellular Matrix
Authors: Jin Xueting , Liu Ying , Addadi Lia , Kruth Howard S
Published: July 2021

We previously reported that cholesterol-enriched macrophages deposit cholesterol into the extracellular matrix and this process depends on ABCA1 and ABCG1. The extracellular cholesterol deposits can be mobilized by HDL, or ApoA-I, the latter dependent on ABCA1-mediated lipidation of ApoA-I. The objective of the current study was to determine the effects of other genes and inhibitors that affect cellular cholesterol trafficking on the deposition of extracellular cholesterol. We used a monoclonal antibody that labels cholesterol microdomains to detect the extracellular cholesterol deposits. Progesterone and U18666A previously were shown to induce accumulation of cholesterol within lysosomes, and we found that these agents also blocked macrophage deposition of extracellular cholesterol. Brefeldin A, an inhibitor of golgi and vesicular trafficking, inhibited extracellular cholesterol deposition. On the other hand, vacuolin, an agent that inhibits the fusion between autophagosomes and lysosomes and thereby induces cellular accumulation of autophagosomes, did not inhibit extracellular cholesterol deposition. To examine the effect of mutant genes known to affect cholesterol trafficking, we examined mutant cultured fibroblasts that were cholesterol-enriched by incubation with LDL plus the LXR agonist, T0901317. Like macrophages, normal cholesterol-enriched fibroblasts deposited extracellular cholesterol that was dependent on ABCA1, because ABCA1-deficient fibroblasts did not deposit extracellular cholesterol. While progesterone and U18666A inhibited fibroblast extracellular cholesterol deposition, surprisingly, fibroblasts deficient in NPC1 or NPC2, which also abnormally accumulate cholesterol within lysosomes during cholesterol enrichment, nevertheless, deposited extracellular cholesterol similar to normal fibroblasts. That lysosomal hydrolysis of LDL cholesteryl ester is involved in extracellular cholesterol deposition was shown by substantially reduced extracellular cholesterol deposition by fibroblasts lacking acid cholesteryl ester hydrolase. Our findings indicate that a pathway not dependent on NPC1 or NPC2 mediates trafficking of cholesterol from lysosomes to the extracellular space.
Abstract 37: Centrosome Destabilization Through the Ubiquitin-Proteasome Pathway Regulates Proplatelet Production
Authors: Machlus Kellie R , Vijey Prakrith , Soussou Thomas , Italiano Joseph E
Published: July 2021

Background:
Proteasome inhibitors such as bortezomib, a chemotherapeutic used to treat multiple myeloma, induce thrombocytopenia within days of initiation. The mechanism for this thrombocytopenia has been tied to data revealing that proteasome activity is essential for platelet formation. The major pathway of selective protein degradation uses ubiquitin as a marker that targets proteins for proteolysis by the proteasome. This pathway is previously unexplored in megakaryocytes (MKs).

Objectives:
We aim to define the mechanism by which the ubiquitin-proteasome pathway affects MK maturation and platelet production.

Results:
Pharmacologic inhibition of proteasome activity blocks proplatelet formation in megakaryocytes. To further characterize how this degradation was occurring, we probed distinct ubiquitin pathways. Inhibition of the ubiquitin-activating enzyme E1 significantly inhibited proplatelet formation up to 73%. In addition, inhibition of the deubiquitinase proteins UCHL5 and USP14 significantly inhibited proplatelet formation up to 83%. These data suggest that an intact ubiquitin pathway is necessary for proplatelet formation.

Proteomic and polysome analyses of MKs undergoing proplatelet formation revealed a subset of proteins decreased in proplatelet-producing megakaryocytes, consistent with data showing that protein degradation is necessary for proplatelet formation. Specifically, the centrosome stabilizing proteins Aurora kinase (Aurk) A/B, Tpx2, Cdk1, and Plk1 were decreased in proplatelet-producing MKs. Furthermore, inhibition of AurkA and Plk1, but not Cdk1, significantly inhibited proplatelet formation in vitro over 83%.

Conclusions:
We hypothesize that proplatelet formation is triggered by centrosome destabilization and disassembly, and that the ubiquitin-proteasome pathway plays a crucial role in this transformation. Specifically, regulation of the AurkA/Plk1/Tpx2 pathway may be key in centrosome integrity and initiation of proplatelet formation. Determination of the mechanism by which the ubiquitin-proteasome pathway regulates the centrosome and facilitates proplatelet formation will allow us to design better strategies to target and reverse thrombocytopenia.
Abstract 371: Deficiency in Microrna-155 Leads to Reduced Atherosclerosis, Increased Obesity and Nonalcoholic Fatty Liver Disease--A Novel Mouse Model of Metabolically Healthy Obesity
Authors: Johnson Candice , Virtue Anthony , Lopez-Pastraña Jahaira , Shao Ying , Fu Hangfei , Li Xinyuan , Li Ya-Feng , Yin Ying , Mai Jietang , Rizzo Victor , Tordoff Michael , Bagi Zsolt , Shan Huimin , Jiang Xiaohua , Wang Hong , Yang Xiao-Feng
Published: July 2021

Metabolically healthy obesity (MHO) describes the phenomenon of overweight and obese patients paradoxically retaining a healthy metabolic profile. The molecular mechanisms underlying MHO remain enigmatic partly due to a dearth of animal models mirroring MHO in patients. Using apolipoprotein E knockout (ApoE
-/-
) mice on high-fat (HF) diet as an atherosclerotic obesity model, we demonstrated: 1) microRNA-155 (miRNA-155, miR-155) is significantly upregulated in aortas of ApoE
-/-
mice; and miR-155 deficiency in ApoE
-/-
mice inhibits atherosclerosis; 2) ApoE
-/-
/miR-155
-/-
(DKO) mice show HF diet-induced obesity, adipocyte hypertrophy and present with nonalcoholic fatty liver disease (NAFLD); 3) DKO mice demonstrate HF diet-induced elevations of plasma leptin, resistin, fed-state and fasting insulin, increased expression of adipogenic transcription factors, but lack glucose intolerance and insulin resistance. Our results are the first to present a metabolically healthy obesity (MHO) model using DKO mice with features of decreased atherosclerosis, increased obesity and NAFLD. Our findings suggest the mechanistic role of reduced miR-155 expression in MHO and present a new MHO working model based on a single miRNA deficiency in diet-induced obese atherogenic mice. Furthermore, our results serve as a breakthrough in understanding the potential mechanism underlying MHO and provide a new biomarker and novel therapeutic target for MHO-related metabolic diseases.
Abstract 377: Mast Cell Serotonin Controls Subcutaneous Adipose Tissue Browning and Systemic Energy Expenditure in Mice
Authors: Zhang Xian , Wang Xin , Shi Guo-Ping , Liu Jian
Published: July 2021

Adipose tissue browning and systemic energy expenditure provide a defense mechanism against obesity and associated metabolic diseases. In western diet-fed mice, inactivation of mast cells (MCs) ameliorates obesity and insulin resistance along with improved metabolic rate. Yet, a direct role of MCs in adipose tissue thermogenesis and browning remains unclear. Here we report that, in the context of mice on a chew diet, norepinephrine (NE)-stimulated metabolic rate is increased in MC-deficient

Kit
w-sh/w-sh

mice and MC-stabilized wide-type (WT) mice. Such functional inactivation of MCs enhances thermogenesis and browning in subcutaneous adipose tissues (SAT), but not in brown (BAT) and epididymal adipose tissues (EAT). MC reconstitution to SAT blocks the aforesaid changes in

Kit
w-sh/w-sh

mice. Mechanistic studies demonstrate that functional inactivation of MCs not only elevates the numbers of PDGFRα
+
bipotential adipocyte precursors but also accelerates beige adipocyte differentiation in SAT. Using tryptophan hydroxylase 1 inhibitor, we show that MC-derived serotonin inhibits SAT beige adipocyte biogenesis and systemic energy expenditure. Together, functional inactivation of MCs or inhibition of MC serotonin synthesis in SAT promotes adipocyte browning and systemic energy metabolism in mice.
Abstract 381: Ideal Cardiovascular Health and Cardiovascular Disease: Heterogeneity Across Event Phenotype and Contribution of Multiple Biomarkers
Authors: Gaye Bamba , Tafflet Muriel , Arveil Dominique , Kee Frank , Evans Alun , Amouyel Philippe , Ferrieres Jean , Empana Jean-Philippe
Published: July 2021

Aims:
To investigate whether or not the association between baseline cardiovascular health (CVH) and incident cardiovascular disease (CVD) differs by event phenotypes and to address the mediating effect of inflammatory and haemostatic blood biomarkers.

Methods:
The association of ideal CVH with outcomes was computed in 9312 middle-aged men from Northern Ireland and France (whole cohort) in multivariable Cox proportional hazards regression analysis. The mediating effect of baseline blood biomarkers was evaluated in a case control study nested within the cohort after 10 years of follow-up.

Results:
After a median follow-up of 10 years, 614 first CHD events and 117 first stroke events were adjudicated. Compared to those with poor CVH, those with an ideal CVH profile at baseline had a 72% lower risk of CHD (HR=0.28; 95% CI: 0.17; 0.46) and a 76% lower risk of stroke (HR=0.24; 95% CI: 0.06; 0.98). No heterogeneity was detected across main CHD and main stroke phenotypes. While significantly lower mean concentrations of hs-CRP, IL-6 (inflammatory markers), and fibrinogen, von Willbrandt factor (haemostatic factors) were noted in the controls with higher CVH status, the association of CVH with incident CHD was not attenuated upon adjustment for these biomarkers.

Conclusion:
these results support the universal promotion of ideal CVH for CVD in general and suggest that the lower risk of CHD associated with ideal CVH is independent from inflammatory and haemostatic biomarkers.
Abstract 383: Patient-Specific Coronary Models Combining Intravascular Ultrasound and Optical Coherence Tomography Lead to More Accurate Plaque Cap Thickness and Stress/Strain Quantifications
Authors: Guo Xiaoya , Monoly David , Yang Chun , Samady Habib , Zheng Jie , Wang Liang , Mintz Gary S , Maehara Akiko , Pei Xuan , Li Zhi-Yong , Tang Dalin , Giddens Don P
Published: July 2021

Accurate cap thickness and stress/strain quantifications are of fundamental importance for vulnerable plaque research. An innovative modeling approach combining intravascular ultrasound (IVUS) and optical coherence tomography (OCT) is introduced for more accurate patient-specific coronary morphology and stress/strain calculations.

In vivo IVUS and OCT coronary plaque data were acquired from two patients with informed consent obtained. IVUS and OCT images were segmented, co-registered, and merged to form the IVUS+OCT data set, with OCT providing accurate cap thickness. Biplane angiography provided 3D vessel curvature. Due to IVUS resolution (150 μm), original virtual histology (VH) IVUS data often had lipid core exposed to lumen since it sets cap thickness as zero when cap thickness <150 μm. VH-IVUS data were processed with minimum cap thickness set as 50 and 180 μm to generate IVUS50 and IVUS180 data sets for modeling use. 3D fluid-structure interaction models based on IVUS+OCT, IVUS50 and IVUS180 data sets were constructed to investigate the impact of OCT cap thickness improvement on stress/strain calculations.

Figure 1 is a brief summary of results from 27 slices with cap covering lipid cores from 2 patients. Mean cap thickness (unit: mm) from Patient 1 was 0.353 (OCT), 0.201 (IVUS50), and 0.329 (IVUS180), respectively. Patient 2 mean cap thickness was 0.320 (OCT), 0.224 (IVUS50), and 0.285 (IVUS180). IVUS50 underestimated cap thickness (27 slices) by 34.5%, compared to OCT cap values. IVUS50 overestimated mean cap stress (27 slices) by 45.8%, compared to OCT cap stress (96.4 vs. 66.1 kPa). IVUS50 maximum cap stress was 59.2% higher than that from IVUS+OCT model (564.2 vs. 354.5 kPa). Differences between IVUS and IVUS+OCT models for mean cap strain and flow shear stress were modest (cap strain: <12%; FSS <2%).

Conclusion:
IVUS+OCT data and models could provide more accurate cap thickness and stress/strain calculations which will serve as basis for plaque research.
Abstract 388: Transcellular Transport of HDL Bearing Gold Nanoparticles Across the Blood Brain Barrier
Authors: Chuang Skylar T , Cruz Siobanth , Stab Julia , Wagner Sylvia , Von Briesen Hagen , Narayanaswami Vasanthy
Published: July 2021

The overall objective of this study was to develop a HDL-based multifunctional platform for transport and delivery of highly hydrophobic gold nanoparticles (AuNP) bearing photothermic properties across the blood brain barrier (BBB). We exploited the ability of apolipoprotein E3 (apoE3) to act as a high affinity ligand for the low-density lipoprotein receptor to gain entry into endothelial and glioblastoma cells. The issue of poor aqueous solubility of AuNP of varying diameters (3, 10, or 10 nm) was overcome by integrating them with phospholipids and apoE3, yielding reconstituted rHDL bearing AuNP (rHDL-AuNP). Transmission electron microscopy (TEM) revealed the presence of AuNP embedded in spherical particles. Incubation of human brain microvasculature endothelial cells or glioblastoma cells with rHDL-AuNP bearing unlabeled or FITC-labeled apoE3 revealed robust uptake of particles that were localized in endocytic/lysosomal vesicles. The transport of rHDL-AuNP across an
in vitro
BBB model developed from primary porcine endothelial cells was examined. The addition of rHDL-AuNP to the luminal side of the cells did not affect the integrity of the BBB as assessed by the localization of key tight junction markers such as occludin, claudins and ZO-1 by immunofluorescence, and, by continual measurement of the transepithelial electrical resistance by impedance spectroscopy under physiological conditions. Lastly, the appearance of fluorescein fluorescence and AuNP in the abluminal side suggested transport of rHDL-AuNP across the neurovascular junction. These findings demonstrate that rHDL bearing apoE3 acts as a detergent in solubilizing and dramatically improving the aqueous solubility of AuNP, facilitates cellular uptake and transcellular transport of rHDL-AuNP across endothelial cells. They are significant since they present rHDL bearing apoE3 as an effective platform for delivering AuNP across the BBB.
Abstract 391: Sterol Efflux Function and Protein Composition of HDL Associates With Recovery From Stroke
Authors: Plubell Deanna , Fenton Alexandra , Wayne Clark , Zakai Neil A , Quinn Joseph F , Alkayed Nabil J , Pamir Nathalie
Published: July 2021

Background:
Prospective cohort studies and meta-analyses examining the relationship between HDL-cholesterol (C) and stroke are discordant and question the value of HDL-C as a marker for stroke risk prediction. Other properties of HDL-C such as cholesterol efflux capacity (CEC) and proteome, are less studied.

Methods:
We investigated the changes in HDL CEC and proteome to determine if they are associated with improved stroke recovery. Plasma from age- and lipid profile-matched healthy controls (N = 35) and stroke patients were collected at 24 (early, N = 35) and 96 hour (late, N = 20) post stroke, and analyzed with three independent assays to measure macrophage-mediated, ABCA1 and ABCG1-specific sterol efflux, and HDL proteome. Stroke recovery was assessed at 3 months using the Modified Rankin Scores (MRS) and the NIH Stroke Scale (NIHSS).

Results:
Both macrophage- and ABCG1-mediated CEC were reduced by 50% (
P
<0.0001) and 20% (
P
<0.038) in early and late post stroke samples, respectively, compared to the control group. Patients who had comparable or increased CEC between the two-time points exhibited lower NIHSS and MRS indicating better recovery. Proteomic analysis of HDL indicated a distinct time-dependent remodeling post stroke. Coagulation complement cascade proteins (FGB, FGA, A2M, C3) significantly increased (FDR>0.01) early and returned to control levels later, inflammation proteins (SAA1, SAA2, PON1, C4B) increased early and continued to increase. Interestingly, platelet adhesion proteins (DSG1, JUP, ITGB1, ITGA2, TUBB, DNAH3, PF4) were abundantly present in only later samples.

Conclusion:
1) patients who maintain or improve HDL CEC post stroke exhibit better recovery scores, 2) post stroke HDL proteome remodeling is dynamic with distinct time-dependent protein signatures that may associate with stroke recovery.
Abstract 398: Bile Acid Receptor Activation Ameliorates Metabolic Disorders by Differentially Activating FXR or TGR5
Authors: Jadhav Kavita S , Xu Yang , Zhang Yanqiao
Published: July 2021

Objectives:
Activation of the bile acid (BA) receptors farnesoid X receptor (FXR) or TGR5 has beneficial effects on metabolic homeostasis. However, activation of FXR may increase obesity and activation of TGR5 has little effect on lipid metabolism. As such, dual activation of FXR and TGR5 appears to be a more attractive approach for treatment of common metabolic disorders. So far, the role of BA receptor activation in metabolic regulation is not well characterized.

Methods:
We utilized wild-type (WT) mice,
Tgr5
-/- mice,
Fxr
-/- mice,
Apoe
-/- mice and
Shp
-/- mice to investigate whether and how BA receptor activation by INT-767, a semisynthetic agonist for both FXR and TGR5, can prevent or reverse diet-induced metabolic disorders.

Results:
INT-767 reversed HFD-induced obesity and hyperglycemia in a TGR5-dependent manner and inhibited the development of atherosclerosis and non-alcoholic fatty liver disease (NAFLD). Mechanistically, INT-767 improved lipid homeostasis by activation of FXR and increased energy expenditure. Furthermore, activation of FXR inhibited several lipogeneic genes in the liver. We identified peroxisome proliferation-activated receptor γ (PPARγ) and CCAAT/enhancer-binding protein α (CEBPα) as novel downstream targets of FXR. FXR inhibited PPARγ expression by inducing SHP (small heterodimer partner) whereas the inhibition of CEBPα by FXR is SHP-independent.

Conclusions:
BA receptor activation can prevent and reverse obesity, NAFLD and atherosclerosis by specific activation of FXR or TGR5. Our data suggest that compared of activation of FXR or TGR5 alone, dual activation of both FXR and TGR5 is a more attractive strategy for treatment of common metabolic disorders.

Key words:
FXR; TGR5; Atherosclerosis; Obesity; NAFLD; Lipogenesis
Abstract 401: Hypercholesterolemia Induces Pro-Inflammatory Changes in Monocytes Which Are Reversed by PCSK9 Antibody Treatment
Authors: Neele Annette E , Bernelot Moens Sophie J , Kroon Jeffrey , van der Valk Fleur M , Van den Bossche Jan , Hoeksema Marten A , Hoogeveen Renate M , Schnitzler Johan G , Baccara-Dinet Marie T , Manvelian Garen , Stroes Erik S , de Winther Menno P
Published: July 2021

Aims:
Migration of monocytes into the arterial wall contributes to arterial inflammation and atherosclerosis progression. Since elevated LDL levels have been associated with activation of monocytes, intensive LDL lowering may reverse these pro-inflammatory changes. Subjects with elevated LDL levels are currently treated with statins, which are also described to have pleiotropic effects. Using proprotein convertase subtilisin/kexin type 9 (PCSK9) monoclonal antibodies which selectively reduce LDL we studied the impact of LDL lowering on monocyte phenotype and function in patients with familial hypercholesterolemia (FH).

Methods and Results:
We assessed monocyte phenotype and function using flow cytometry for a broad range of monocyte-relevant markers and a trans-endothelial migration assay in FH patients (n=22: LDL-C 6.8±1.9mmol/L) and healthy controls (n=18, LDL-C 2.9±0.8mmol/L). Interestingly, monocyte chemokine receptor (CCR) 2 expression was approximately 3-fold increased in FH patients compared with controls (ΔMFI 605±214 vs 236±155
P
<0.001). CCR2 expression correlated significantly with plasma LDL-C levels (r=0.709) and positively associated with intracellular lipid accumulation. Monocytes from FH patients also displayed enhanced migratory capacity
ex vivo.
After 24 weeks of PCSK9 monoclonal antibody treatment (n=17), plasma LDL-C was reduced by 49% (from 6.7±1.3 mmol/L to 3.4±1.3 mmol/L
P
<0.001), which coincided with reduced monocyte intracellular lipid accumulation and suppressed CCR2 expression (ΔMFI: baseline 607±209, post PCSK9 mAbs: 207±180,
P
<0.001). Functional relevance was substantiated by the reversal of enhanced migratory capacity of monocytes following PCSK9 monoclonal antibody therapy. All changes were comparable in subjects who were treated with statins (n=14: LDL-C 2.8±0.6mmol/L) indicating that the anti-inflammatory effects were mainly mediated through LDL lowering.

Conclusions:
Elevated LDL levels in FH induce pro-inflammatory changes in monocytes, which is dampened by PCSK9 monoclonal antibody therapy. LDL lowering was paralleled by reduced intracellular lipid accumulation, suggesting that LDL lowering itself is associated with anti-inflammatory effects on circulating monocytes.
Abstract 405: Synthetic Low Density Lipoprotein Receptor Knockout Mouse Model to Study Atherosclerosis Regression
Authors: Basu Debapriya , Hu Yunying , Mullick Adam E , Graham Mark J , Barnhart Shelley , Fisher Edward A , Bornfeldt Karin E , Goldberg Ira J
Published: July 2021

Regression of atherosclerotic plaques occurs when plasma total cholesterol (TC) is markedly reduced. Commonly used animal models of regression include a surgical model of transplantation of atherosclerotic arteries from

Apoe
-/-

or

Ldlr
-/-

mice to C57BL/6J mice, and the ‘Reversa mouse’ in which hypercholesterolemia due to deletion of LDL receptor (LDLR) and over-expression of apoB100 is conditionally reversed. These models require either challenging surgeries or time-consuming breeding strategies. We utilized a synthetic method to create and then reverse hypercholesterolemia and atherosclerosis by transient knockdown of hepatic LDLR using antisense oligonucleotides (ASOs). C57BL6/J mice on Western diet were treated once a week for 16 weeks with intraperitoneal injections of LDLR ASO. TC increased to ~600 mg/dl in two weeks, and remained high. After 16 weeks, one group of mice was sacrificed for baseline analysis and the remaining mice were treated with the sense ASO to antagonize any residual LDLR ASO activity. The mice were then either kept on Western diet or switched to chow diet. Within a week, TC decreased to ~150 mg/dl in both regression groups and mice were sacrificed after another week. LDLR mRNA and protein analysis in the baseline group showed that ASO-mediated LDLR deletion was efficient. Similar analysis in both regression groups at the end of the study confirmed recovery of hepatic LDLR expression. Mice in the baseline group had visible plaques in the aortic arch and brachiocephalic artery (BCA). Analysis of the BCA showed that lesion size, visualized by Movat’s staining, was not significantly different between the baseline and regression groups. BCA lesions stained for MAC-2, a marker for macrophages, showed reduced macrophage content in both regression groups but more significantly in the Western diet group. Our data show that lesion remodeling and regression, in terms of inflammatory cell content, occur in our model. These changes occur relatively quickly as we analyzed lesions after two weeks of plasma cholesterol reversal. In conclusion, we have developed a synthetic LDLR hepatic knockout mouse model of atherosclerosis regression that can be used for any genetically modified mouse strain and obviates the need for extensive mouse breeding.
Abstract 408: Enzyme Modified LDL Induces Migration and Calcification of Human Coronary Artery SMCs
Authors: Chellan Bijou , Hofmann Bowman Marion
Published: July 2021

Background:
Enzyme modified LDL (ELDL) is present in human atherosclerotic lesions and is a major foam cell-forming modified LDL for murine vascular smooth muscle cells (SMC) as reported by us previously. Here we study ELDL and its effects on human coronary artery SMC (HCASMC) in vitro.

Methods and Results:
Incubation of HCASMC with 10 μg/ml ELDL (trypsin, cholesterol esterase modified) resulted in significant foam cell formation (analyzed by Oil Red O, lipid measurement) compared to HCASMC incubated with oxidized LDL (200 μg/ml; -75%, p<0.01) or native LDL (200 μg/ml; -50%, p<0.01). Whole genome gene expression (Illumina Bead Chip HT12v4, analyzed by DAVID v6.8 and IPA) of HCASMC treated with ELDL, oxLDL, LDL, and control (cell culture medium only) showed several top canonical pathways specifically induced by ELDL, together with activated upstream regulators including p38MAPK, NFkB. ERK. Upregulation of ANGPTL4 and BMP-2 -mRNA (22 and 2 fold respectively over native LDL) was verified by qRT-PCR and immunoblotting. ELDL-induced foam cells showed dose dependent (1-20 μg/ml ELDL) increase in migration in collagen coated trans well dishes, which was attenuated by Lacidepine, a known inhibitor of ELDL uptake in murine SMCs. Furthermore, rANGPTL4 also upregulated HCASMC migration dose dependently (1-5 μg/ml for 24 h) and was comparable with the migration induced by ELDL. However, Lacidipine had no effect on rANGPLT4 mediated migration, suggesting that ANGPLT-4 independently of ELDL uptake promotes migration of HCASMC. In calcification assays using MEM with 0.2% FCS and 1.5 mM phosphate, ELDL at 2.5 μg/ml induced more calcification native LDL (>25%, p<0.01, analyzed by alizarin red staining and organic extraction, and this was proceeded by increase in BMP-2 mRNA.

Conclusions:
ELDL is highly potent in inducing foam cells in cultured HCASMC. Whole genome expression and bioinformatics analysis indicate up-regulation of pathways linked to osteochondrogenic transformation. BMP-2 and ANGPTL4 are significantly upregulated in ELDL-induced HCASMC foam cells. These results point to the potential of ELDL to induce migratory and osteoblastic effects in HCASMC with potential implications in SMC migration and calcification in human atherosclerosis.
Abstract 410: Inhibiting Mitochondrial Fission in Progressive Atherosclerosis Protects Against Ischemia/Reperfusion Injury
Authors: Duggan Simon
Published: July 2021

Introduction:
Two opposing highly regulated mitochondrial processes, division (fission) and fusion, determine cell-type specific mitochondrial morphology, intracellular distribution and activity. Mitochondrial dysfunction has been implicated in atherogenesis and in cardiac ischemia/reperfusion (I/R) injury.

Hypothesis:
We hypothesize that increased mitochondrial fission plays a role in progressive atherosclerosis and protection in I/R injury.

Methods and Results:
Male apolipoprotein E knockout mice (ApoE-/-) were fed either a high-fat diet (21% lard & 0.15% cholesterol - HFD) or chow diet for 24 weeks from weaning. Compared to their chow fed littermates, mice fed a HFD demonstrated smaller, shorter and reduced density of left ventricular (LV) interfibrillar mitochondria suggestive of mitochondrial fission by electron microscopy. Furthermore, there was decreased expression of the mitochondrial fusion proteins, optic atrophy 1 and mitofusin 1 and increased fission protein Fis 1. Whilst the proteins Mfn2 and Drp1 were unchanged. On ischemia, LV tissue expression of fission proteins was increased in both groups. In isolated perfused hearts subjected to 30 min ischemia and 120 min reperfusion inhibition of the Drp1-mediated fission with Mdivi-1 (25 μM) at the onset of reperfusion significantly reduced infarct size (n=6/group). Inhibition of the DRP1-mediated fission at serine 637 in langendorff hearts conferred relatively more cardio-protection in the chow fed mice compared to western diet (19.93% ± 2.219%, n=5 versus 29.61% ± 2.517%, n=6; p < 0.05). The reduction in myocardial necrosis was corroborated by a significantly reduced release of creatine kinase and recovery of coronary flow.

Conclusion:
Inhibiting mitochondrial fission protects hearts against ischemia/reperfusion injury in progressive atherosclerosis.
Abstract 416: Targeted Echogenic Liposome Binding Forces Correlate With Atheroma Ultrasound Image Enhancement
Authors: Klegerman Melvin E , Laing Susan T , Kim Hyunggun , Kee Patrick H , Moody Melanie R , Huang Shao-Ling , McPherson David D
Published: July 2021

Background:
We have developed echogenic immunoliposomes (Ab-ELIP) as a means of highlighting atheroma at different stages of atherogenesis. We have devised methods to measure conjugated antibody (Ab) binding affinity (targeting efficiency, TE) in order to assess and predict Ab-ELIP imaging enhancement and therapeutic efficacy. We have now constructed an algorithm for calculating binding force from the evaluation data and have tested it relative to ultrasound (US) image enhancement results.

Hypothesis:
One or more Ab-ELIP binding force parameters is predictive of ultrasound imaging enhancement of atheroma in vivo.

Methods:
Ab-ELIP were prepared by conjugating specific MAbs to ELIP. Conjugation efficiency (CE) was then determined by a quantitative immunoblot assay and particle enumeration with a Beckman-Coulter Multisizer to yield CE in molecules Ab/liposome. Conjugated Ab affinity (K
D
and K
assoc
) was derived from ELISA data and used to generate specific TE (CE x relative binding area) and functional avidity (K
assoc
x specific TE). The functional avidity was then converted to free energy of association using the equation of state (ΔG = -RT
ln
K). Using specific TE, ΔG in kcal/mole was converted to binding energy in erg/liposome, from which binding forces, E
b
, in dyne/liposome were derived. Based on specific TE and Ab molecules per m
2
, binding force/liposome was converted to Pascal (10 dyne/cm
2
). Finally, dyne/liposome was converted to piconewton (pN)/molecule, a common measure of binding force.

Results:
Using rabbit and miniswine atherosclerotic models, we previously demonstrated US imaging enhancement of atheroma by MAbs specific for ICAM-1, α
v
β
3
-integrin and VCAM-1 conjugated to ELIP. Percent image enhancement correlated (p < 0.05) with binding force in Pascal, but not in dyne/liposome, indicating the importance of binding area to TE. Binding force in pN/molecule ranged from 311 to 385, which agrees well with the published results of others.

Conclusions:
We have discovered a binding force parameter calculated from CE and TE data that is predictive of Ab-ELIP targeting performance in vivo. The next step, to demonstrate a similar correlation with therapeutic efficacy, will then provide an important tool for clinical Ab-ELIP optimization.
Abstract 418: Co-Culture of Endothelial Cells, Smooth Muscle Cells and Monocytes in Collagen Scaffold: A Novel i
n vitro
Model of Atherosclerosis
Authors: Liu Martin , Karagiannis Angelos , Sis Matthew , Kidambi Srivatsan , Chatzizisis Yiannis
Published: July 2021

Objectives:
To develop and validate a 3D
in-vitro
model of atherosclerosis that enables direct interaction between various cell types and/or extracellular matrix.

Methods and Results:
Type I collagen (0.75 mg/mL) was mixed with human artery smooth muscle cells (SMCs; 6x10
5
cells/mL), medium, and water. Human coronary artery endothelial cells (HCAECs; 10
5
/cm
2
) were plated on top of the collagen gels and activated with oxidized low density lipoprotein cholesterol (LDL-C). Monocytes (THP-1 cells; 10
5
/cm
2
) were then added on top of the HCAECs. Immunofluorescence showed the expression of VE-cadherin by HCAECs (A, B) and α-smooth muscle actin by SMCs (A). Green-labelled LDL-C particles were accumulated in the subendothelial space, as well as in the cytoplasm of HCAECs and SMCs (C). Activated monocytes were attached to HCAECs and found in the subendothelial area (G-I). Both HCAECs and SMCs released IL-1β, IL-6, IL-8, PDGF-BB, TGF-ß1, and VEGF. Scanning and transmission electron microscopy showed the HCAECs monolayer forming gap junctions and the SMCs (D-F) and transmigrating monocytes within the collagen matrix (G-I).

Conclusions:
In this work, we presented a novel, easily reproducible and functional
in-vitro
experimental model of atherosclerosis that has the potential to enable
in-vitro
sophisticated molecular and drug development studies.
Abstract 419: Macrophage Dicer Expression is Required for VEGF-A-induced Arteriogenesis
Authors: Mantsounga Chris , Healy Abigail L , Ceneri Nicolle , Morrison Alan R
Published: July 2021

During ischemia, inflammatory arteriogenesis is determined by macrophage VEGF-A expression. A number of microRNA’s have been identified to downregulate VEGF-A and VEGF-mediated angiogenic processes. We sought to determine the microRNA(s) that were most critical to regulating macrophage VEGF-A expression by genetically deleting Dicer in myeloid cells. In a hindlimb ischemia model of angiogenesis and arteriogenesis, we found reduced rescue of hindlimb perfusion in macrophage

Dicer
-/-

animals. Surprisingly, this was associated with decreased macrophage VEGF-A production and leaky, dysfunctional arteries as evidenced by microCT angiograms, indicating that Dicer expression and consequent microRNA biogenesis are required for appropriate VEGF-A expression and VEGF-mediated arteriogenesis. The mechanism of reduced macrophage VEGF expression involved decreased VEGF-A mRNA stability with decreased association of VEGF-A mRNA with the RNA-stabilizing protein HuR. Our results support a paradigm shift in the perception of Dicer expression and microRNA biogenesis as a negative regulator of VEGF-A expression and implicate microRNA biogenesis in promoting macrophage VEGF-directed arteriogenesis in response to ischemia. Identification of this novel mechanism of VEGF-A regulation has strong implications in the development of targeted therapeutic strategies that can promote appropriate arteriogenesis in the setting of obstructive vascular disease or inhibit inappropriate arteriogenesis in the setting of malignant oncological diseases.
Abstract 420: Focal Adhesion Kinase Disrupts Nuclear Factor-kB Signaling and Alleviates Atherogenesis
Authors: Murphy James , Lim Steve
Published: July 2021

Introduction:
While we have discovered that focal adhesion kinase (FAK) is critical for global pro-inflammatory gene expression, it remains unclear what role FAK plays in vascular inflammation. Our preliminary data suggested that FAK might regulate a major inflammatory signaling factor, NF-kB. The goals of this study are to investigate the mechanisms underlying FAK-mediated inflammatory gene expression in endothelial cells (ECs), and the effect of FAK inhibition on atherogenesis.

Hypothesis:
FAK activity is required for pro-inflammatory gene expression via NF-kB activation in ECs.

Methods:
Human aortic ECs (HAoECs) were treated with TNF-α and with/without a FAK inhibitor (FAK-I). Inflammatory gene expression and NF-kB signaling was evaluated via western blot, immunocytochemistry or qRT-PCR. To access vascular inflammation
in vitro
, primary mouse monocytes were used in an adhesion assay on TNF-α stimulated HAoECs. To evaluate the importance of FAK activity in atherogenesis, partial carotid artery ligation was performed in ApoE-/- mice. Mice were fed a high fat/high cholesterol diet and were given either FAK-I or vehicle by oral gavage for two weeks. Both carotid arteries were harvested and analyzed.

Results:
We found that FAK activity is required for expression of various pro-inflammatory molecules such as VCAM-1 and MCP-1, and FAK inhibition blocks sustained NF-kB activation. Mechanistically, while FAK inhibition does not block NF-kB activation after 1hr TNF-α stimulation, NF-kB activity significantly reduced after 3hr when compared to TNF-α only. This decrease in NF-kB activity was correlated with a decrease in NF-kB nuclear localization and an increase in IkB protein stability. FAK inhibition reduced monocyte attachment to TNF-α stimulated HAoECs (3.8 vs 22.1 fold). Mice treated with FAK-I showed decreased VCAM-1 expression, macrophage infiltration and lipid accumulation compared to vehicle group.

Conclusion:
These results suggest that FAK is critical for global inflammatory gene expression and plays a key role in maintaining sustained NF-kB activity during chronic inflammation in ECs. FAK inhibitors may prove useful as an “anti-inflammatory drug” in the treatment of cardiovascular disease.
Abstract 421: Computational Modelling Suggests That Reversing Low HDL May Not Always Lead to Plaque Regression
Authors: Myerscough Mary R , Chalmers Alexander D
Published: July 2021

Introduction:
Clinical evidence suggests that high numbers of high density lipoprotein (HDL) particles in the bloodstream and high levels of HDL efficacy, reduce the risk of heart attack and stroke. Experimental evidence suggests that increasing HDL particle number too late in the life of the plaque does not lead to regression but only to reduced rates of plaque growth.

Hypothesis:
We postulate that nonlinear dynamics are intrinsic to how cells respond to HDL in reverse cholesterol transport (RCT) from macrophages and foam cells and that these dynamics have consequences for plaque growth, regression and the plaques’ responses to changes in the plaque environment, such as changes in low density lipoprotein (LDL) influx.

Methods:
We formulated a computational model for the endothelium and the inside of the artery wall. This model consisted of six partial differential equations and associated boundary conditions. We found what conditions led to model plaques remaining small and what led to model plaques growing unboundedly. We also determined the outcomes if the concentration or action of HDL changes at different times in the life of the model plaque.

Results:
Model plaques with high concentrations of HDL, highly efficacious HDL and low levels of LDL were small and did not grow. There was a tipping point (or bifurcation point) so that plaques with HDL concentrations below the tipping point grew unboundedly and plaques with HDL concentrations above the tipping point remained small. The position of this tipping point depended on the concentration of modified LDL experienced by the plaque, anti-inflammatory actions of HDL and the phenotypes of macrophages after RCT. Increasing HDL concentration after the model plaque had grown led to plaque regression only if the dynamics of the plaque were such that the model plaque was able to shrink to a small plaque that did not grow. If the reduction in HDL was too late, or not large enough, the model plaque could not regress and continued to grow.

Conclusion:
The nonlinear nature of the dynamics of plaque growth and regression may result in outcomes to treatment that seem unexpected or inexplicable. Computational modelling provides a means of exploring the consequences of these dynamics.
Abstract 423: Telomerase Deficiency in Hematopoietic Stem Cells Decreases Atherosclerosis by Silencing STAT3-Dependent Inflammation in Macrophages
Authors: Qing Hua , Heywood Elizabeth , Jones Karrie L , Doyon Genevieve , Bruemmer Dennis
Published: July 2021

Objective:
Telomerase Reverse Transcriptase (TERT), the catalytic subunit of telomerase, supports critical cellular responses required for tissue remodeling. Previous studies established that TERT expression is induced in activated macrophages and during experimental and human atherosclerosis formation. In the present study, we investigated the role of TERT for atherosclerosis development and macrophage inflammation.

Approach and Results:
TERT-deficient mice were crossbred with LDL-receptor-deficient (LDLr-/-) mice to generate first generation G1TERT-/-LDLr-/- offsprings, which were then further intercrossed to obtain third generation G3TERT-/-LDLr-/- mice. G1TERT-/-LDLr-/- mice revealed no telomere shortening while severe telomere attrition was evident in G3TERT-/-LDLr-/- mice. When fed an atherogenic diet, G1TERT-/-LDLr-/- and G3TERT-/-LDLr-/- mice were both protected from atherosclerosis formation compared to their wildtype controls, indicating that genetic TERT-deletion prevents atherosclerosis, and formation of the disease is not affected by telomere attrition. Similarly, atherosclerosis development was decreased in chimeric LDLr-/- mice with TERT deletion in hematopoietic stem cells after bone marrow transplantation. TERT deficiency reduced macrophage accumulation in atherosclerotic lesions and altered chemokine expression, including CXC1/2/3, CCL3, CCL5, CCL21, CCR7, IL-6, and IL-1α. In isolated macrophages, sequence analysis of silenced inflammatory gene promoters indicated that TERT deletion altered signal transducer and activator of transcription 3 (STAT3)-dependent chemokine expression and recruitment of phosphorylated STAT3 to its target promoters. Mechanistically, TERT expression was necessary and sufficient to maintain STAT3 phosphorylation. Finally, we demonstrate that TERT deletion induces transcription of PIAS and SOCS3, both are endogenous inhibitors of STAT3 pathway.

Conclusions:
We propose that genetic TERT deficiency decreases atherosclerosis formation by silencing inflammatory chemokine transcription through inactivation of the STAT3 signaling pathway.
Abstract 428: Role of Thrombospondin-1 in Atherosclerotic Complications Associated With Metabolic Syndrome
Authors: Sahu Soumyadip , Ganguly Rituparna , Rea Brianna , Cerroni Abigail , Mathias Amy , Raman Priya
Published: July 2021

Atherosclerosis is the leading cause of increased morbidity and mortality in metabolic syndrome (MetS), a constellation of risk factors that include hyperglycemia and visceral obesity. Individuals with MetS have increased incidence of vascular smooth muscle cell (VSMC) migration and proliferation, a key characteristic of VSMC phenotypic transition. We previously reported that high glucose and high leptin, mimicking hyperglycemia and obesity, independently upregulate a potent proatherogenic matricellular protein, thrombospondin-1 (TSP-1), expression in VSMCs. The goal of the present study was to investigate the role of TSP-1 in development of atherosclerosis in MetS. We generated a mouse model of combined MetS and atherosclerosis (
KKAy
+/-
/ApoE
-/-
) by crossing obese hyperglycemic agouti yellow
KKAy
+/-
mice with atherosclerotic ApoE
-/-
mice. Upon weaning, yellow
KKAy
+/-
/ApoE
-/-
mice and age-matched black
KKAy
-/-
/ApoE
-/-
littermates were maintained on regular chow diet from 4-18 wks of age. Mice were euthanized after an overnight fasting; blood, aortae and heart were collected for lipid profiling, immunoblotting and atherosclerotic lesion studies. Yellow
KKAy
+/-
/ApoE
-/-
mice showed significant increase in body weight, blood glucose and plasma lipid levels vs. black
KKAy
-/-
/ApoE
-/-
littermates. Aortic root morphometry demonstrated increased lesion burden (Oil red O) and neointimal thickening (H & E) in MetS
KKAy
+/-
/ApoE
-/-
vs. non-MetS
KKAy
-/-
/ApoE
-/-
mice. Immunohistochemistry further revealed co-localization of α-SMA (SMC marker) and PCNA (proliferation marker) expression in aortic roots of
KKAy
+/-
/ApoE
-/-
vs.
KKAy
-/-
/ApoE
-/-
mice. Notably, lesion formation was associated with an increase in TSP-1 expression in aortic vessels. Consistently, TSP-1 and PCNA expression were elevated in aortic SMC (aSMC) primary cultures derived from obese diabetic

KKAy
+/-

mice vs. lean non-diabetic
KKAy
-/-
littermates. Finally, incubation of aSMC with a TSP-1 blocking peptide reduced PCNA and vimentin (SMC synthetic marker) expression coupled with attenuated SMC proliferation in obese diabetic

KKAy
+/-

mice. Together, these results suggest a putative role of TSP-1 in accelerated atherosclerosis and VSMC phenotypic transition in MetS.
Abstract 435: An Electronic Health Records Query for Severe Hypercholesterolemia Identifies Individuals With Undiagnosed Clinical and Molecular FH at a Tertiary Academic Centre
Authors: Ajufo Ezim , Ueda Masako , Sikora Tracey , deGoma Emil M , Dilzell Kristen , Raper Anna , DerOhannessian Stephanie , Hossain Erik , Mucksavage Megan L , Cuchel Marina , Rader Daniel J
Published: July 2021

Background:
Familial hypercholesterolemia (FH) is a common heritable disorder of elevated low density lipoprotein cholesterol (LDL-C) with an estimated prevalence of 1/200-300 in the US; however, fewer than 10% of cases have been identified. We wanted to examine whether a simple EHR query for severe hypercholesterolemia could be used to identify clinically and genetically defined cases of FH.

Objectives/Purpose:
We tested the hypothesis that querying the EHR using an LDL-C criterion would be a novel way to screen for and ultimately identify undiagnosed cases of FH.

Methods:
An EHR screening query was used to identify active adult patients with LDL-C ≥ 220 mg/dL in the University of Pennsylvania outpatient EHR database. Patients with secondary causes of hypercholesterolemia and those who had previous genetic testing for FH were excluded. The query identified 3,475 individuals, 120 were subsequently consented and enrolled for molecular testing. This was performed with next-generation sequencing using Progenika’s SEQPRO LIPO IS platform, targeting
LDLR, APOB, PCSK9
and
LDLRAP1
. A literature search was performed to gather information on identified
LDLR
variants of unknown significance (VUS). In addition,
in-silico
analysis was employed to evaluate the pathogenicity of the
LDLR
and
LDLRAP1
VUS.

Results:
Among the 120 subjects, 53 (44.2%) met the Dutch Lipid Clinic Network (DLCN) criteria for probable or definite clinical FH. Molecularly, 19 FH-related pathogenic mutations were found in 18 (15%) individuals. Four had a common
APOB (
R3500Q) mutation, 14 had a
LDLR
mutation. One individual had a double heterozygous mutation in
PCSK9
and
LDLR.
In addition, 17
LDLR
VUS were identified in 16 (13.9%) individuals. A literature review and
in-silico
analysis predicted that 8 VUS found in 10 subjects were “disease causing”. Therefore, a total of 28 (24.3%) subjects from our cohort carried either a FH causal mutation or likely pathogenic variant. Overall, 59 subjects (49.2%) in our cohort were ascertained to have either a clinical or molecular diagnosis of FH.

Conclusion:
The use of an EHR screening query for severe hypercholesterolemia was a novel, low investment but relatively high yield approach for identifying undiagnosed cases of FH at a tertiary academic centre.