bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 336: Overrepresentation of Familial Hypercholesterolemia-causing Mutations in Patients with Familial Combined Hyperlipidemia
Authors: Bamimore Mary A , Cao Henian , Wang Jian , McIntyre Adam , Johansen Christopher , Hegele Robert A
Published: March 2022

Background_

Elevated plasma low-density lipoprotein (LDL) cholesterol is a feature of both familial hypercholesterolemia (FH) and familial combined hyperlipidemia (FCH). FH is predominantly a monogenic _autosomal dominant_ condition that results from mutations in the genes coding for low density lipoprotein receptor (LDLR), apolipoprotein B-100 (APOB) and proprotein convertase subtilisin/kexin type 9 (PCSK9). In contrast, the genetic basis of FCH, a very common dyslipidemia in the human population, is less well understood, but is considered to be polygenic. Investigations in other polygenic lipid disorders indicate that genetic susceptibility is comprised of both common polymorphisms and rare heterozygous mutations. Re-sequencing technologies have identified greater accumulation of missense rare variants in candidate genes of diseased cohorts relative to healthy controls, thus indicating a potential role of candidate genes in disease etiology.

Objective_

Our aim was to understand the genetic etiology of FCH. We hypothesized missense rare variants in the 3 known autosomal dominant FH-causing genes and the newly characterized inducible degrader of the low density lipoprotein receptor (IDOL), would accumulate in FCH patients (n=138) (cases) relative to controls (n=94). We tested this hypothesis using Sanger sequencing of coding sequences, intron-exon boundaries and some regulatory regions of the 4 candidate genes.

Results & Discussion_

For cases vs controls, the total missense rare variant carrier counts were 7/138 vs 4/94 in LDLR and 14/138 vs 9/94 in APOB. Of all these missense rare variants found in our entire cohort, we found functionally verified FH-causing variants only in our cases and not in our controls (P =0.09), namely LDLR G314S, D333V, V806I and APOB R3500W variants. These variants likely explain hypercholesterolemia in those particular FCH patients.

Conclusions_

This preliminary analysis of FCH candidate gene sequences supports the idea that rare FH-causing mutations are over-represented in FCH. The findings also support future expanded case-control and family studies to identify possible monogenic causes of FCH using various next-generation sequencing technologies.
Abstract 34: Myh11 R247C is a Modifier Allele for Aortic Disease
Authors: Kwartler Callie , Kuang Shao-Qing , Prakash Siddharth , Huang Jian , Kamm Kristine , Stull James , Sweeney Lee , Milewicz Dianna
Published: March 2022

Thoracic aortic aneurysms and dissections (TAAD) affect more than 30,000 Americans annually. TAAD can be inherited due to mutations in a number of genes, including MYH11, which encodes the smooth muscle myosin heavy chain. In non-familial TAAD patients, hypertension is the most common risk factor, but genetic contributors have not been explored. Exome sequencing has identified non-synonymous variants of unknown significance in MYH11 in the population, and these variants are enriched in TAAD patients without a family history. The goal of this study was to determine if rare MYH11 variants contribute to TAAD.

A knock-in mouse model of one recurrent rare variant, Myh11 R247C, shows no structural or pathologic abnormalities of the aorta, but does have decreased aortic contractility. We sought to determine if the Myh11 R247C allele could modify aortic disease due to another gene mutation. The Myh11R247C allele was bred into Acta2-/- mice, which develop aortic aneurysms, and into Acta2+/- mice, which do not. In both cases, the R247C allele significantly increases aortic diameter and accelerates elastin fragmentation and proteoglycan deposition; however the aneurysms do not progress to aortic dissection.

Treatment of Myh11R247C/R247C mice with a combination of L-NAME and high salt diet induced a sustained increase in systolic blood pressure from 130mmHg to 180mmHg. Approximately 25% of Myh11R247C/R247C mice on treatment died spontaneously with evidence of aortic dissections at necropsy, but without aortic dilation. The surviving mice also show no aneurysm formation. Aortic pathology shows significant fragmentation and thinning of elastin fibers, medial thickening, and a unique pattern of proteoglycan deposition along the elastin layers in the hypertensive Myh11R247C/R247C mice.

These data indicate that the Myh11R247C allele in mice increases severity of Acta2-/- associated aortic aneurysms and induces aortic disease when added to a known risk factor for TAAD, hypertension. Therefore, patients with the Myh11R247C rare variant and a disease-causing mutation may have earlier disease onset and increased severity; co-occurrence of the Myh11R247C allele in individuals with other benign genetic variants or risk factors like hypertension may induce TAAD.
Abstract 344: A20 Increases Endothelial Nitric Oxide Synthase Expression and Activity to Sustain Endothelial Cell Function under Inflammatory Insults
Authors: Chien Victor , Kaczmarek Elzbieta , Daniel Soizic , Essayagh Sanah , Lee Andy , Choi Lynn Y , Choi Jean Y , Da Silva Cleide G , Ferran Christiane
Published: March 2022

Nitric oxide generated by endothelial nitric oxide synthase (eNOS) acts at different levels to sustain proper endothelial cell (EC) function. Pro-inflammatory molecules associated with various pathological conditions negatively affect eNOS expression and activity, altering vascular homeostasis. Decreased eNOS levels and activity are the pathognomonic features of EC dysfunction and a prelude to atherosclerotic remodeling of the vasculature. We have demonstrated that A20 maintains EC homeostasis by inhibiting NF-kappa B activation and protecting from apoptosis. The aim of this study is to explore whether A20 impacts eNOS expression/activity.

A20 was overexpressed in human coronary artery EC (HCAEC) by recombinant adenovirus-mediated gene transfer. Effects of A20 on eNOS expression and phosphorylation were analyzed by quantitative PCR, human eNOS promoter-luciferase reporter assay, chromatin immunoprecipitation (ChIP) and Western blot. Our data demonstrate that over-expression of A20 in HCAEC significantly increased eNOS mRNA and protein levels by promoting eNOS transcription, as demonstrated by ChIP, and eNOS promoter analysis using Luciferase reporter assays. Importantly, A20-induced upregulation of eNOS expression was sustained in HCAEC treated with tumor necrosis factor that inhibits eNOS transcription and function. Moreover, A20 enhanced phosphorylation of eNOS (Ser-1177), a surrogate marker of its activity, by promoting the activation of its upstream kinase AKT.

Our data demonstrate that A20 upregulates and activates eNOS, even in EC exposed to inflammation. A20’s ability to promote and sustain eNOS activity could be critical to its cytoprotective effect in EC. Further, it supports our pursuit of A20-based therapies to maintain vascular homeostasis, preventing vascular diseases such as atherosclerosis, transplant arteriosclerosis and diabetic vasculopathy.
Abstract 348: Fibrin Targeted in vivo Imaging of Tissue Plasminogen Activator-induced Thrombolysis of Mouse Deep Vein Thrombosis
Authors: Stein Ashley F , Kessinger Chase W , Zalada Castro Henry , Erdem S S , McCarthy Jason R , Lin Charles P , Jaffer Farouc A
Published: March 2022

Introduction

Tissue plasminogen activator (tPA) is increasingly used to treat deep vein thrombosis (DVT) to prevent the post-thrombotic syndrome. However, while aging venous thrombi are likely to become resistant to fibrinolysis, the age-dependent response to fibrinolysis has not been systematically assessed. Here we used confocal intravital microscopy (IVM) to dynamically study fibrinolysis of murine thigh DVT at high-resolution using thrombus angiography and a fibrin-specific sensor, FTP11-CyAm7 (FTP11). We hypothesized that fibrinolysis could be tracked and quantified
in vivo
using serial IVM and molecular imaging of fibrin.

Methods

C57Bl/6 mice underwent topical 7.5% ferric chloride injury to induce femoral DVT. At day 1 (n=6) or day 4 (n=5) post DVT, IVM was performed during tPA infusion. Mice were i.v. injected with a bolus of tPA, heparin and plasminogen, followed by a continuous 1 hour infusion of tPA+heparin. FTP11, injected 45 min prior to lysis, enabled molecular imaging of fibrin. DVT area and length were quantified using FITC-dextran venography before and after lysis. IVM image stacks were analyzed by NIH ImageJ. After sacrifice, fluorescence microscopy (FM) and Carstair’s staining were performed.

Results

Compared to day 4, day 1 DVT fibrinolysis showed a greater net decrease in thrombus area (32±11 vs. 6±4%, p=0.004) and FTP11 signal (54±17 vs. 11±8%, p=0.004) after 60 min. Of note, day 1 more lyseable DVT had significantly higher baseline FTP11 signal (target-to-background ratio 1.91±0.86 vs. 1.10±0.06, p=0.004), consistent with greater thrombus permeability at day 1 vs. day 4. The net lysis correlated well with both the baseline FTP11 thrombus signal (r=0.62, p=0.048) and change in FTP11 signal (r=0.79, p=0.006). IVM angiography revealed a trend of increased lysis in the distal thrombus edge, compared to the middle or proximal edge (day 1: 44±11% vs. 26±16% and 29±26%, p>0.05). FTP11 signal colocalized with fibrin-rich thrombi on FM and Carstair’s staining.

Conclusion

Chemically induced murine DVT lyse more effectively at day 1 than day 4, as tracked by serial IVM molecular imaging. The FTP11-CyAm7 fibrin signal differentiates DVT of different ages and informs the magnitude of triggered fibrinolysis.
Abstract 358: Association of Adiponectin-with Cardio-metabolic Traits
Authors: Khan Hassan , Kunutsor Setor , Steur Marinka , Haycock Philip , Gregson John
Published: March 2022

Background and Objectives

Current evidence on the association of adiponectin with fatal or non-fatal cardiovascular (CVD) outcomes is inconsistent. In addition association between adiponectin and related metabolic traits has not been systematically evaluated.

Design and Methods

A systematic search was performed using MEDLINE and Web of Science databases. Effect estimates and standard errors of associations of adiponectin with metabolic traits including (adiposity, glucose, insulin and cholesterol) and cardiovascular outcomes (coronary heart disease and stroke) were extracted from relevant prospective studies. The effect estimates and standardized correlation coefficients were pooled together using random effects meta-analysis.

Results

A total of 18 studies with 32,116 participants and 7,433 cardiovascular events reported on associations for adiponectin. Moderately strong and inverse associations were observed for adiponectin and measures of adiposity, body mass index, r=-0.13 [95% CI -0.29 to -0.02], waist to hip ratio, (r=-0.25 [95% CI -0.31 to -0.21] and waist circumference, r=-0.23 [95% CI -0.29 to -0.16]. Similarly inverse associations were seen with glucose, r=-0.19 [95% CI -0.22 to -0.17], insulin, r=-0.27 [95% CI -0.38 to -0.15] and triglycerides r=-0.31[95% CI -0.36, -0.26] while a positive correlation was observed with HDL-c, r=0.35[95% CI 0.30-0.41]. A pooled adjusted RR[95% CI] of 0.99 (95% CI: 0.86 to 1.14) with CVD for individuals in the top versus bottom third of baseline levels of adiponectin was seen. There was evidence of moderate heterogeneity between the findings of contributing studies (I2 =66%) with little difference explained by several study-level characteristics. Similarly modest associations were observed for both coronary heart disease (CHD), RR[95% CI] 0.89 (0.74 to 1.08) and stroke RR[95% CI] 1.01 (0.83 to 1.22).

Conclusion

Results of this systematic analysis give further evidence to the role of adiponectin with metabolic traits. However observational evidence from prospective studies indicates modest association with CHD and stroke outcomes that needs further investigation.
Abstract 359: Effects of Erythritol on Endothelial Function in People with Type 2 Diabetes Mellitus
Authors: Flint Nir , Hamburg Naomi M , Decock Peter , Bosscher Douwina , Vita Joseph A
Published: March 2022

Erythritol is a polyol compound used as a low calorie nutritive sweetener. Several animal model studies have shown that erythritol has antioxidant properties and may improve endothelial function. The goal of this pilot study was to obtain evidence for such an effect in patients with type 2 diabetes mellitus (DM). Vascular testing included brachial artery flow-mediated dilation (FMD), reactive hyperemia, digital peripheral arterial tonometry (EndoPAT), carotid-femoral pulse wave velocity (PWV), central pulse pressure, and brachial blood pressure. We also measured blood glucose, insulin, C-peptide, C-reactive protein and urine F2 isoprostanes. Twenty-four participants (age 56±5 yrs, 54% female) consumed erythritol as a flavored beverage (12g x3/day) for 28 days. We assessed the effect of erythritol on vascular function compared to baseline at three time points: Acute (2 hours after erythritol 24 g), chronic (at day 28), and acute-on-chronic (day 28 after erythritol 24g). We did not observe a significant change in brachial artery FMD or reactive hyperemia. Acute consumption of erythritol produced a significant improvement in endothelial function measured by EndoPAT (lnPAT ratio 0.52±0.48 vs. 0.87±0.29, P=0.005). lnPAT ratio was 0.72±0.33 (P=0.19 vs. baseline) after chronic consumption and 0.84±0.34 after acute-on-chronic consumption (P=0.02). Following 28 days of erythritol consumption we observed a favorable effect on markers of aortic compliance: significant reduction in central pulse pressure from 47±13 to 41±9 mmHg (P=0.021) and strong trend for decreased PWV from 9.1±1.4 to 8.6±1.6 cm/sec (P=0.06). Markers of glucose metabolism, oxidative stress and inflammation did not vary throughout the study. We observed a trend for reduced systolic blood pressure after chronic erythritol consumption (131±16 to 128±11 mmHg, P=0.10). A subgroup analysis of 12 patients with systolic blood pressure above 130 mmHg yield a significant reduction in systolic blood pressure from 144±12 to 133±9 mmHg (P=0.01). This pilot study suggests that erythritol may have favorable effects on small vessel endothelial function, blood pressure, and central aortic stiffness in DM patients. Confirmation of these findings will require a placebo-controlled study.
Abstract 378: Global MicroRNA Evaluation of The Brains and Aortas of a Prenatal Stress Murine Model With Vulnerable Plaque Show Sexual Dimorphism
Authors: Jhamnani Sunny , Aghili Nima , Lassance-Soares Roberta , Sood Subeena , Devaney Joseph , Burnett Mary Susan , Epstein Stephen
Published: March 2022

Introduction and Hypothesis

Stress is a major cardiovascular risk factor. Preliminary studies in our lab demonstrated that prenatal stress in a murine model is associated with accelerated atherosclerosis and a vulnerable plaque phenotype at 20 weeks of age. Here, we hypothesized that prenatal stress is associated with global microRNA (miRNA) changes, possibly gender specific, and we sought to determine them by seeking such changes in two different tissues (brain and aorta) in both sexes.

Methods

Pregnant mixed background female mice were divided into stressed (chronic cold stress for the last 17 days of gestation) and non-stressed groups. Prenatally stressed and non-prenatally stressed offspring had urine corticosteroid levels measured at six weeks to see the effects of stress. At eight weeks the offspring were sacrificed and global miRNA levels were measured using the Affymetrix 2.0 miRNA chip on the brains and aorta.

Results

Urine corticosteroid levels were decreased in both genders at six weeks (p≤0.01), which has been shown in previous severe stress murine models. These were more significant in males (p=0.006) than in females (p=0.01). In eight-week-old males, there were 84 miRNAs in the brain that were dysregulated between prenatally stressed and non-stressed (p<0.05). The top miRNAs in males by p-value were miR-26, miR-21, and miR-346. Females that were eight weeks old had 89 dysregulated miRNAs (p<0.005) in the brain with miR-154 and miR-331 as the top miRNAs by p-value. Analysis of the aortas revealed 74 and 92 dysregulated miRNAs (p<0.05) with miR-326 and miR-15 as the top hits in males and females by p-value, respectively. Of interest, these top miRNAs have, in prior published studies, been implicated in playing a role in ischemia, ischemic preconditioning, stress and endothelial degeneration.

Conclusions

Prenatal stress leads to variety of miRNA changes that persist into at least the eighth postnatal week of life. These alterations in miRNA levels may contribute to the development of a vulnerable plaque phenotype that we have shown to be present at 20 weeks of age in a previous study. Prenatal stress also appears to exert gender specific effects, a finding that may provide insight into gender differences in cardiovascular outcomes.
Abstract 392: Effects of Dietary Omega-3 Polyunsaturated Fatty Acids Plus Minus Aspirin on Plasma Lipids and Hepatic Gene Expression in ApoE-Deficient Mice
Authors: Sorokin Alexander , Vaisman Boris , Thacker Seth , Remaley Alan
Published: March 2022

BACKGROUND

Omega-3 fatty acids (O3FA) have hypolipemic, anti-inflammatory and atheroprotective effects. Aspirin (ASA) may potentiate these beneficial effects, by altering the production of anti-inflammatory mediators, such as resolvins.

OBJECTIVE

To compare the anti-atherogenic effects and gene expression changes of O3FA diet plus minus ASA.

METHODS

12-week old apoE-Ko females were placed for 2 weeks on a O3FA-deficient (O3FA-) diet, and were then treated for 13 weeks with either O3FA-, O3FA+ or O3FA+ASA diet (N=10). RNA from liver (N=4) was isolated and analyzed by Qiagen PCR Array for 84 genes related to cholesterol metabolism. Arteriosclerosis was assessed by
en face
analysis.

RESULTS

O3FA+ diet decreased plasma total cholesterol, triglycerides, phospholipids, free cholesterol and cholesteryl esters between 26%-39% (
P
<0.0005). Majority of the decrease in cholesterol was due to a lowering of VLDL-C and LDL-C. Similar lipoprotein changes were observed in mice on the O3FA+ASA diet. O3FA+ fed mice showed numerous significant changes in the expression of genes related to cholesterol metabolism. Insig1 was decreased by 1.9-fold, whereas Nr1h4 and Prkaa1 were increased by 1.5 fold. Cholesterol biosynthesis genes, such as Dhcr7, Fdps and Hmgcr were decreased in 1.6, 2, and 1.4-fold, respectively. Expression of Acaa2 and Hmgcs2, genes involved in catabolism of cholesterol, were increased by 1.7-1.8 fold. O3FA+ASA treated mice showed a similar hepatic gene expression profile except PCSK9, which decreased 5.9-fold in these mice but only by 2.5-fold in O3FA+ treated mice. Compared to O3FA- treated control mice, O3FA+ASA treated mice showed 2.2-fold decrease in aortic atherosclerosis, whereas no change compared to control mice was observed with just the O3FA+ diet.

CONCLUSIONS

Addition of ASA to the O3FA+ diet showed a greater effect in lowering the expression of PCSK9 in the liver and decreased aortic atherosclerosis. ASA treatment, however, did not show a significant further improvement in the lowering of plasma pro-atherogenic lipoproteins over that observed with the O3FA+ diet, suggesting that the anti-inflammatory property of ASA may account for its anti-atherogenic effect.
Abstract 402: HDL Mimetic Peptides ATI-5261 and CS-6253 Induce ABCA1 Oligomerization, Cellular Cholesterol Efflux and Generate Nascent HDL-Like Lipoproteins
Authors: Hafiane Anouar , Bielicki John , Johansson Jan , Genest Jacques
Published: March 2022

Novel approaches that target HDL biogenesis may increase nascent HDL-like particle formation that can be used for therapeutic purposes. Here, we evaluated two 26-amino acid apoA-I mimetic peptides designed from c-terminus of apoE, ATI-5261 and CS-6253 in their ability to promote cellular cholesterol efflux via the ABCA1 transporter. Using mifepristone-inducible ABCA1 BHK cells and cAMP-stimulated J774 macrophages, we examined ABCA1 oligomerization with dithiobis[succinimidyl propionate] (DSP) as a cross linker. Like native apoA-I, ATI-5261 and CS-6253 induced ABCA1 oligomerization at equimolar concentrations, and appear to interact with native oligomers of ABCA1, similar to apoA-I, as judged by crosslinking revealed by SDS-PAGE. By sucrose density gradient fractionation, we show that ABCA1 oligomerization occurs in non-raft domains. ATI-5261 and CS-6253 promoted cellular cholesterol efflux in a dose-dependent saturable manner. In BHK cells, the efficiency of cholesterol efflux (Km) on an equimolar basis was: apoA-I (0.16±0.02), ATI-5261 (0.45±0.08) and CS-6253 (0.68±0.15) and the efflux capacity (Vmax) (% in 24h) was: apoA-I (14.54±0.61), ATI-5261 (11.98±0.51) and CS-6253 (10.87±0.66), indicating a higher efflux efficiency for the apoA-I mimetics but with less capacity than native apoA-I. A similar trend was observed in J774 cells. Desorption of cellular cholesterol at 45 min occurred in both non-raft and raft-like membrane domains. We then examined by HPLC and 2-dimentional PAGGE the size and types of HDL-like particles formed. In 3[H]-cholesterol loaded BHK-ABCA1 cells, incubation with ATI-5261 and CS-6253 for 45 min and 12 hours yielded α-migrating particles with an apparent size ranging between 9 and 17 nm, as revealed by specific antibodies directed against ATI-5261 and CS-6253. Taken together, these observations suggest that ATI-5261 and CS-6253 induce cellular cholesterol efflux via ABCA1 oligomerization at molar concentrations similar to apoA-I and with higher efficiency. The particles generated by ATI-5261 and CS-6253 have α-migrating mobility with a size ranging between 9 and 17 nm and contain cholesterol, similar to nascent HDL particles.
Abstract 41: Integrin Outside-in Signaling Regulates Local Microenvironments Within Hemostatic Plugs
Authors: Stalker Timothy J , Welsh John D , Wu Jie , Brass Lawrence F
Published: March 2022

Prior studies have demonstrated that platelet activation within a hemostatic plug is heterogeneous. In particular, we recently determined that following vascular injury in vivo, a platelet mass is composed of distinct regions defined by the extent of platelet activation and other properties. At least two regions are apparent: a core of fully activated and degranulated platelets immediately adjacent to the site of injury, which is overlaid by a shell of less activated platelets that have not undergone a-granule secretion. Here, we tested the hypothesis that platelet accumulation and activation are regulated by the interaction of platelet signaling pathways with local microenvironments present within the different regions of a hemostatic plug using novel confocal intravital imaging approaches. We also hypothesized that integrin outside-in signaling plays a role in shaping the core region microenvironment. The results reveal that plasma volume (i.e. porosity) and solute transport velocity are significantly reduced in the interplatelet space of a hemostatic plug as compared to the bulk blood flow following laser-induced injury in mouse cremaster arterioles. Further, porosity and solute transport are significantly reduced in the core region of the hemostatic plug as a result of tight platelet packing, as compared to the outer shell region composed of loosely adherent platelets. To examine the impact of integrin outside-in signaling we utilized mice lacking beta 3 integrin cytoplasmic tail tyrosine phosphorylation (diYF). Prior studies have shown unstable platelet aggregation and defective fibrin clot retraction in diYF mice. We found diYF mice formed smaller hemostatic plugs following vascular injury in vivo. This decrease in total platelet accumulation was associated with reduced formation of the hemostatic plug core region and decreased fibrin generation, indicating decreased thrombin activity within the hemostatic plug. Taken together, these results support the hypothesis that tight platelet packing provides a protected microenvironment within a hemostatic plug in which bioactive molecules may accumulate to support platelet activation. They also identify a novel role for aIIbb3 outside-in signaling in hemostatic plug formation.
Abstract 410: Binding of Apolipoprotein A-I to Inner and Outer Components of Bacterial Membranes
Authors: Beck Wendy H , Adams Christopher P , Biglang-awa Ivan M , Weers Paul M
Published: March 2022

Human apolipoprotein A-I (apoA-I) is a 28 kDa protein and a major component of high-density lipoproteins, mediating several essential metabolic functions related to heart disease. The protein is able to suppress bacterial growth of gram-negative bacteria. To understand the basis of the bacterial growth suppression and its potential as an antimicrobial protein, the binding interaction between apoA-I and membrane components of gram-negative bacteria was characterized using wild-type, modified, and a deletion variant of apoA-I. To modify the protein, lysine residues were acetylated with acetic anhydride. The deletion variant was created by removing the C-terminal amino acid residues 190-143. The binding interaction of apoA-I with lipopolysaccharides (LPS), abundantly present in the outer bacterial membrane of
E. coli
, was investigated with non-denaturing PAGE and isothermal titration calorimetry. In addition, protein structural changes induced by LPS binding were monitored by circular dichroism and fluorescence spectroscopy. Binding analysis showed that wild-type and N-terminal apoA-I were able to associate with LPS, but the acetylated protein showed a strongly reduced binding interaction. The bacterial inner membrane is rich in negatively charged phosphatidylglycerol. ApoA-I showed a strong preference for bilayer vesicles made of phosphatidylglycerol compared to phosphatidylcholine. Acetylated apoA-I showed a marked decrease in phosphatidylglycerol binding, while the binding activity of N-terminal apoA-I was only moderately decreased. These results indicated that apoA-I interacts with components of the inner and outer membrane of gram-negative bacteria. In addition, lysine side-chains play a crucial role in the binding interaction, indicating the importance of ionic interactions. These results indicate the potential for apoA-I to function as an antimicrobial protein.
Abstract 465: Heparanase Alters Endothelial Phenotype, Leukocyte Attachment and Atherosclerotic Plaque Formation
Authors: Johnson Collin G , Bondada Vidya , Yeager Doug , Emelianov Stanislav , Baker Aaron
Published: March 2022

Little is known about the role of enzymatic regulation of glycosaminoglycans in the formation of atherosclerotic plaques. We tested the hypothesis that heparanase (HPA) could regulate endothelial cell inflammatory responses and immune cell mobilization, promoting atherogenesis.

We transduced human endothelial cells with lentiviral vectors for one of four constructs expressing shRNA targeted to HPA or a scrambled control sequence. These shRNA knocked down HPA expression 30-50% of wild type levels. We also overexpressed HPA in these cells using lentiviral constructs. Finally, we transduced cells to express miRNA 1258 known to reduce levels of HPA in endothelial cells. These cell lines were confirmed for gene modulation using PCR and western blotting for HPA, then assayed for the expression of inflammatory markers such as ICAM, VCAM, and IL-8 under static and physiologic levels of shear stress (12 dynes/cm
2
). Increasing HPA levels led to a corresponding increase in inflammatory cytokines in endothelial cells, while reduction of HPA levels by targeted shRNA or miRNA caused reduced expression. Using fluorescently labeled monocytes we exposed the endothelial cell layer to circulating leukocytes in an in-vitro flow system. Adhesion of monocytes increased in cell lines with enhanced HPA expression and decreased in those with HPA knockdown.

We also evaluated the effects of low endothelial sheer stress in transgenic mice overexpressing the human HPA gene crossed with the ApoE knockout mouse line. We surgically implanted an extravascular cuff on the left carotid artery of mice from this HPA-Tg/ApoE line and the ApoE line to create a region of disturbed flow and low shear stress. We used high resolution combined ultrasound and photoacoustic imaging system to image the flow and plaque morphology after 1 day, 2 weeks and 4 weeks post-implantation. Doppler imaging revealed overexpression of HPA increases the formation and severity of atherosclerosis. The oscillatory flow in the carotid artery due to the cuff placement lead to increased plaque accumulation and increased inflammation in response to the induced shear stress in the HPA-Tg/ApoE mice compared to the ApoE control mice.

Our studies support HPA as a potent regulator of inflammation and plaque formation.
Abstract 475: Cationic Peptides in Inflammation and Atherosclerosis
Authors: Selvarajan Krithika , Aluganti Narasimhulu Chandrakala , Parthasarathy Sampath
Published: March 2022

Background

Apoprotein (Apo A1) and apoprotein E mimetic peptides are known to reduce atherosclerosis. Antioxidant, anti-inflammatory, and hypolipidemic properties have been suggested to contribute to the anti-atherosclerotic effects. In this study, we tested whether three distinct and unrelated cationic peptides would inhibit inflammatory response to lipopolysaccharide (LPS) and oxidized low density lipoprotein (Ox-LDL).

Methods

5F-mimetic peptide of apoA1, LL27 derived from the anti-microbial peptide CAMP, and a human glycodelin derived peptide were commercially synthesized. RAW 264.7 cells were incubated with LPS or Ox-LDL, pretreated with and without peptides. RNA was isolated from treated cells and real time PCR was performed using mouse IL-1α and IL-6 primers.

Results

Increased IL-1α and IL-6 gene expression was observed in the presence of LPS/Ox-LDL. However, peptides showed dual effect as they induced inflammatory markers in the presence of LPS except for LL27 but all peptides inhibited the Ox-LDL-induced inflammatory gene expressions. The results suggest that these peptides may have dual effects on inflammatory cytokine genes in the presence of LPS and Ox-LDL.

Conclusion

Based on these results, we predict that lysine-rich cationic peptides could have therapeutic potential in reducing CVD/atherosclerosis-associated inflammation. However, specific receptors, presence of lipids, and other factors may influence their effects.
Abstract 546: Negative Regulation of Reactive Oxygen Species From Nadph Oxidases by Caveolar Proteins
Authors: Chen Feng , Yu Yanfang , Fulton David
Published: March 2022

The importance of caveolar proteins, including caveolin-1 (Cav1) have been widely explored in cardiovascular disease. Cav1 binds to numerous signaling proteins via interactions with its scaffolding domain, and caveolin-2 (Cav2) and Cavin1 depend on Cav1 for stability and expression. The NADPH oxidases (Nox) are a primary source of reactive oxygen species (ROS) and oxidative stress. Loss of cav1 has been shown to increase vascular ROS production and can promote cardiovascular diseases such as pulmonary arterial hypertension. However, the ability of Cav1 to regulate Nox enzymes is not known. Here, we found that increased expression of Cav1 by gene transfer or by administration of its scaffolding domain peptide, potently inhibited ROS production from Nox1, Nox2, Nox4 and Nox5. In contrast, inhibition of endogenous Cav1 with small interfering RNA increased ROS production from human vascular cells. The Cav1 scaffolding domain mutant, Cav1 F16A, failed to inhibit Nox activity. In addition, Cav2 and Cavin-1 can also decreased ROS production from Nox enzymes. To further explore the underlying mechanism, we found Cav1 did not modify intracellular calcium but reduced ROS output in an isolated enzyme assay. Furthermore, we found increased Cav1, Cav2 and Cavin-1 expression robustly decreased the expression level of Nox proteins. Given the similar intracellular location of Cav1 and Nox and the presence of highly conserved Cav1 binding sequence in Nox enzymes, we predict that caveolin proteins may directly bind with Nox and promote their degradation. It is also possible that degradation is mediated by Cav1 regulated Nox protein ubiquitylation. Together, these results suggest that caveolin proteins negatively regulate NADPH oxidases to ameliorate cellular stress and are an important regulatory pathway in the cardiovascular system.
Abstract 60: Targeted Disruption of LDLR Causes Hypercholesterolemia and Atherosclerosis in Yucatan Miniature Pigs
Authors: Swart John , Davis Bryan , Wang Xiao-Jun , Rohret Judy , Struzynski Jason , Merricks Elizabeth , Bellinger Dwight , Rohret Frank , Nichols Timothy , Rogers Christopher
Published: March 2022

Recent progress in engineering the genomes of large animals has spurred increased interest in developing better animal models for diseases where current options are inadequate. Here, we report the creation of Yucatan miniature pigs with targeted disruptions of the low-density lipoprotein receptor (LDLR) gene in an effort to provide an improved model of hypercholesterolemia and atherosclerosis. Yucatan miniature pigs are well established as translational research models because of similarities to humans in physiology, anatomy, genetics, and importantly, size. Using gene targeting and somatic cell nuclear transfer, male and female LDLR+/- pigs were generated. Subsequent breeding of heterozygotes produced LDLR-/- pigs. When fed a standard swine diet, LDLR+/- pigs exhibited a moderate, but consistent increase in total and LDL cholesterol, while LDLR-/- pigs had dramatically elevated levels. Furthermore, this severe hypercholesterolemia in the LDLR-/- pigs resulted in atherosclerotic lesions in the coronary arteries and abdominal aorta that resemble human atherosclerosis. LDLR-targeted Yucatan miniature pigs offer several advantages over existing models including size, consistency, availability, and cost. This new model of cardiovascular disease could be a transformative resource for developing and testing novel detection and treatment strategies for coronary and aortic atherosclerosis and its complications.
Abstract 7: Targeted Mutagenesis of Antithrombin III in Zebrafish Results in Disseminated Intravascular Coagulation and Thrombosis
Authors: Liu Yang , Richter Catherine , Maeder Morgan L , Vo Andy , Huarng Mike , Swaroop Alok , Joung J.Keith , Shavit Jordan A
Published: March 2022

Pathologic blood clotting (thrombosis) is a leading cause of morbidity and mortality in the developed world, underlying deep vein thrombosis, myocardial infarction, and stroke. Identification of small molecule modifiers using an in vivo thrombosis would facilitate novel and improved therapeutics for patients. The zebrafish is a powerful genetic model in which the hemostatic system is nearly entirely conserved with mammals and its ability to generate thousands of offspring at low cost makes it a powerful tool to screen chemical modifiers of coagulation disorders. We have generated a zebrafish model of complete antithrombin III (at3) deficiency by targeted mutagenesis using zinc finger nucleases. at3 heterozygotes with 22 and 90 base pair deletions (at3[[Unable to Display Character: ∆]]22 and at3[[Unable to Display Character: ∆]]90) were intercrossed, and no homozygotes have survived beyond 7 months of age. Offspring were genotyped at various time points revealing a significant reduction of expected homozygotes compared to the expected Mendelian frequency as early as 1-2 months post fertilization. This lethality was rescued by an at3 cDNA transgene, enabling survival to at least 15 months of age. Analysis of adult homozygous mutants revealed evidence of large intravascular thrombi and externally visible secondary hemorrhage. We induced thrombosis at 3-4 days post fertilization in at3[[Unable to Display Character: ∆]]90 mutant larvae by laser injury and injection of bovine thrombin. Contrary to our expectations, we observed diminished rates of cardinal vein occlusion in homozygous mutants, and this could be rescued by an at3 cDNA transgene. We hypothesized that this was due to disseminated intravascular coagulation (DIC), and observed reduced fibrinogen levels in at3 homozygous mutant plasma. Furthermore, injection of mutant larvae with human fibrinogen prior to laser injury rescued the mutant phenotype. In conclusion, loss of At3 protein results in subclinical DIC in zebrafish larvae secondary to unopposed thrombin activity, but they survive and succumb to lethal thrombosis in adults. Future studies will adapt this model for high throughput screening for genetic and chemical modifiers of DIC and thrombosis. Genes and small molecules identified will be potential novel diagnostic and therapeutic modalities for patients with these disorders.
Abstract 77: Proprotein Convertase Subtilisin/kexin Type 9 (PCSK9) Targets the CD36 Receptor for Degradation
Authors: Demers Annie , Lauzier Benjamin , Des Rosiers Christine , Mayer Gaetan
Published: March 2022

The scavenger receptor CD36 is a membrane glycoprotein with broad ligand specificity that is highly expressed in metabolic tissues. Major functions of CD36 include the uptake of long chain fatty acids in fat tissue, heart and skeletal muscle and the uptake of oxidized lipoproteins in macrophages. Recent findings have also demonstrated that this protein regulates adipogenesis and adipocyte lipolysis. Thus, CD36 plays important roles in energy utilization from fatty acids and in lipoprotein and lipid homeostasis. However, dysfunction of CD36 has been linked to glucose intolerance, diabetes, atherosclerosis, arterial hypertension and cardiomyopathy. Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a secreted glycoprotein that regulates plasma low-density lipoprotein (LDL) cholesterol metabolism by promoting degradation of the hepatic LDL receptor. Here, we report that PCSK9 also induces the degradation of the CD36 receptor. Both cellular expression and secreted exogenous PCSK9 strongly downregulated the levels of CD36 protein in several cell lines. In addition, immunofluorescence analysis using the HepG2-hCD36 cell line demonstrated that the overexpression of PCSK9 decreases the levels of CD36 in parallel with a decrease in the uptake of oxidized LDL. Furthermore, treatment of 3T3-L1 adipocytes with purified soluble PCSK9 protein resulted in a decrease in CD36 protein expression at the cell surface and in the total cellular pool in a dose-dependent manner. Interestingly, we showed that CD36 degradation is modulated by PCSK9 in a proteasome- and lysosome-dependent manner. Co-immunoprecipitation assays in HEK293 cells confirmed CD36 ubiquitination in presence of PCSK9 and also revealed that PCSK9 associates in a complex with CD36. These results support a potential role for PCSK9 to impact the function of CD36 in lipid metabolism.
Abstract 8: TMEM16f Regulates PhosphatidylSerine (PS) Exposure and PS+ Microparticle Generation From Mouse Platelets
Authors: Mukaz Debora K , Fowajuh Ann-Desdemonia N , Li Dongjun , Caplan Jeff , Vortkamp Andrea , Woulfe Donna
Published: March 2022

Transmembrane (TMEM)16f is a protein from the family of TMEM16 Ca++-dependent Chloride channels that is essential for optimal Ca2+-dependent phospholipid scrambling activity in platelets. Recently, it has been shown that TMEM16f-dependent exposure of phosphatidylserine (PS) on platelet surfaces is required for platelet-dependent pro-coagulant activity and hemostasis. However, relatively little is known about the effect of TMEM16f on platelet signaling, functional activity, and microparticle formation. TMEM16f-/- mice were generated in the laboratory of Andrea Vortkamp and bred in our laboratory as heterozygous crosses. When assessed by flow cytometry, platelets from TMEM16f-/- mice exposed 27.8% of the PS exposed by WT platelets in response to thrombin and convulxin, 23.1% of the PS in response to 1 uM
A23187
, and 38% of the PS exposed by WT platelets in response to 10 uM
A23187.
Similar trends were obtained using high resolution immunofluorescent microscopy (TMEM16f-/- exposed 49.2% of the PS detected on WT platelets in response to thrombin + convulxin; 25.6% PS in response to 1 uM
A23187
). Microparticle generation from TMEM16f-/- platelets compared to WT platelets was also evaluated by both high resolution immunofluorescent microscopy and flow cytometry. The proportion of PS+αIIb+ microparticles generated per platelet was lower in platelets from TMEM16f-/- mice compared to WT when evaluated by both of these methods. Given that TMEM16f-/- platelets elicit similar calcium responses upon thrombin and convulxin exposure to those of WT platelets, these data may suggest that PS scrambling activity is required not only for PS exposure on the outer leaflet of platelet membranes, but also for the shedding of PS+ microparticles. Surprisingly, TMEM16f-/- platelets were less sensitive to AYPGKF-induced fibrinogen binding than WT platelets (n= 4, p<.05). Finally, TMEM16f-/- mice form less stable occlusive thrombi following 10% ferric chloride injury of the carotid artery (0/4 stable thrombi of TMEM16f-/-) than WT mice (7/9 stable thrombi). The loss of procoagulant microparticles may contribute to the lower sensitivity of TMEM16f-/- mice to ferric chloride-induced thrombosis.
Abstract 82: Macrophage Specific ABCA1 Deletion Does Not Worsen Insulin Resistance in High Fat Diet Induced or Genetic Obese Mouse Models
Authors: Zhu Xuewei , Bi Xin , Chuang Chia-Chi , Chung Soonkyu , Brown Amanda L , Cuffe Helen , Gebre Abraham K , Boudyguina Elena , Parks John S
Published: March 2022

Introduction

Obesity associated low-grade, chronic inflammation underlies the pathogenesis of type 2 diabetes and insulin resistance. ATP-binding cassette transporter A1 (ABCA1), a membrane lipid transporter, forms nascent HDL particles by effluxing cellular free cholesterol (FC) and phospholipids to lipid-poor apolipoprotein A-I. In macrophages, ABCA1 also dampens pro-inflammatory MyD88-dependent Toll-like receptor signaling by reducing cellular membrane FC and lipid raft content, indicating a role for ABCA1 in macrophage-mediated innate immunity. However, the role of ABCA1 in macrophage-associated innate immunity in insulin resistance is unclear.

Hypothesis

We tested the hypothesis that macrophage-specific ABCA1 deletion may exacerbate insulin resistance by increasing obesity-induced chronic, low-grade inflammation.

Methods and Results

Wild type (WT) and macrophage-specific ABCA1 knockout (MSKO) mice in the C57/BL6 background were fed a 45% High Fat Diet (HFD) for 16-24 weeks or crossed into the leptin deficient (ob/ob) background to induce obesity. Our results showed that MSKO and WT mice developed obesity, insulin resistance, hypercholesterolemia, and hepatic steatosis to a similar extent with HFD feeding or after crossed into the ob/ob background. Resident peritoneal macrophages from HFD-fed or leptin deficient MSKO vs. WT mice accumulated significantly more cholesterol. Relative to chow, HFD markedly induced macrophage infiltration and inflammatory cytokine expression to a similar extent in adipose tissues in WT and MSKO mice. Among the examined pro-inflammatory cytokines in peritoneal macrophages, only IL-6 was highly up-regulated in ob/ob MSKO vs. WT control mice, indicating that lack of ABCA1 in macrophages does not significantly affect obesity associated chronic inflammation.

Conclusion

Although macrophage specific ABCA1 deficiency enhances Toll like receptor signaling, it does not significantly exacerbate obesity associated low grade, chronic inflammation, therefore has no impact on the pathogenesis of insulin resistance in both HFD induced and genetic obese mouse models.
Abstract 99: The Effect of Androgen Deprivation Therapy on Conduit Artery Endothelial Function in Men with Prostate Cancer
Authors: Zuflacht Jonah , Milian Jessica , Kadivar Samoneh , Graham Powell , Nguyen Paul , Beckman Joshua A
Published: March 2022

Introduction

Androgen deprivation therapy (ADT) is a standard treatment for patients with aggressive prostate cancer. While ADT improves cancer-specific and overall survival, it can increases insulin resistance and the risk of diabetes. Emerging evidence suggests that ADT also increases the incidence of adverse cardiovascular events in patients with cardiovascular disease, but the exact mechanism is unknown.

Hypothesis

We hypothesized that ADT may impair endothelial function due to increases in insulin resistance and provide a mechanism for heightened cardiovascular risk in this population.

Methods and Results

We prospectively evaluated conduit artery function in 10 consecutive men (mean age 65 ± 7 years; 30% with diabetes) with prostate cancer before and 3 months after initiation of ADT. High-resolution B-mode ultrasound was used to assess flow-mediated (endothelium-dependent) and nitroglycerine-mediated (endothelium-independent) brachial artery vasodilation. Endothelium-dependent vasodilation was greater at 3 months compared with baseline (11.9 ± 1.7% vs. 9.23 ± 1.8%, p = 0.002). There was no significant change in resting arterial diameter, reactive hyperemic stimulus, mean arterial pressure, nor heart rate between study conditions. Nitroglycerine-mediated, endothelium-independent vasodilation did not change from baseline (18.3 ± 3.55%) to the three month visit (19.7 ± 2.40%), p = 0.466). Markers of insulin resistance will be available at the time of the meeting.

Conclusions

In contrast to our expectation, ADT improves endothelial function. This suggests that the physiological mechanism linking ADT with increased cardiovascular events is not likely regulated by the vascular endothelium. Moreover, this mechanism of insulin resistance may be beneficial to endothelial function.
Abstract 104: EBI3 Deficiency Exacerbates Experimental Abdominal Aortic Aneurysms
Authors: Iida Yasunori , Tanaka Hiroki , Hu Xiaolei , Xiong Xiaoxing , Zhao Heng , Xu Baohui , Dalman Ronald L
Published: March 2022

Background:
Abdominal aortic aneurysms (AAAs) are an age-and gender-related chronic vascular disease. Ebi3 (EB virus-induced gene 3) encodes a cytokine subunit protein shared by two anti-inflammatory cytokines IL-27 and IL-35. Given the importance of inflammation in AAA pathogenesis, we evaluated the influence of Ebi3 deficiency on experimental AAAs.

Methods and Results:
AAAs were created in10 week old male Ebi3-knoctout (KO) and wild type (WT) mice via the intra-aortic infusion of porcine pancreatic elastase (PPE), and evaluated using
in vivo
transabdominal ultrasonography and histology at sacrifice (Figure). Following PPE infusion, KO mice experienced more significant and rapid aneurysmal aortic enlargement. All KO and WT mice developed AAAs (≥50% diameter increase) within 7 and 14 days, respectively. Medial elastin and smooth muscle cell attenuation were more pronounced in aneurysmal aortae of KO mice, as were both aortic macrophage and T cell infiltration. However, no difference in aortic B cell infiltration or mural angiogenesis was noted between groups.

Conclusions:
Ebi3 deficiency promotes the onsets and progression of experimental AAAs in association with increased aortic accumulation of macrophages and T cells. Our study suggests a protective role of Ebi3 in experimental AAAs, potentially related to IL-27 and/or IL-35 production.