bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 361: Epistasis Analysis Identifies Extracellular Matrix Genes Interacting With the COL4A1/COL4A2 Coronary Artery Disease Locus
Authors: Turner Adam , Nikpay Majid , Lau Paulina , Soubeyrand Sebastien , McPherson Ruth
Published: July 2021

The COL4A1/COL4A2 locus on chromosome 13q34 was significantly associated with coronary artery disease (CAD) in the CARDIoGRAM (Nature Genetics, 2011) and the CARDIoGRAMPlusC4D (Nature Genetics, 2013) meta analyses of several large genome-wide association studies (GWAS). Determination of causative SNPs stemming from GWAS data requires a number of bioinformatic and lab based approaches. Functional analysis of GWAS loci can be aided by epistasis analysis, which interrogates synergistic associations with a given trait between pairs of SNPs, either at the same locus or at different loci. We investigated 4 CAD GWAS cohorts in this study (Ottawa Heart Genomics Study A, Ottawa Heart Genomics Study B, Cleveland Clinic Gene Bank, Duke CATHGEN Study). For epistasis analysis we tested whether CAD-associated SNPs at the COL4A1/COL4A2 locus displayed interaction with other CAD-associated SNPs across the genome and used 2 lists of SNPs. The first list of SNPs were ones at the COL4A1/COL4A2 locus significant for CAD association and the second list of SNPs were ones significantly associated with CAD from across the genome. Using PLINK we then generated a list of SNP pairs showing interaction (Pinteraction<0.05) for CAD association within 1 or more cohorts. Epistasis analysis generated over a dozen loci that demonstrated interaction with COL4A1/COL4A2 SNPs for CAD association, many of which have putative functional roles with respect to CAD. Genes integral to pathways involving COL4A1/COL4A2 were of particular interest in understanding the role of type IV collagen in CAD pathogenesis. We identified several extracellular matrix genes showing interaction with COL4A1/COL4A2 including fibronectin (FN1), COL18A1 as well as transmembrane proteins including ITGA2, encoding integrin α2 and SERPINF1, a member of the serpin peptidase family that strongly inhibits angiogenesis. Other interesting loci include NOTCH4, genes involved in angiogenesis, and mediators of TGFβ signalling. Functional studies are currently being conducted to decipher the roles of COL4A1/COL4A2 and these interacting extracellular matrix genes in CAD.
Abstract 37: Secretion of von Willebrand Factor by Endothelial Cells Links Salt to Hypercoagulability and Thrombosis
Authors: Dmitrieva Natalia I , Burg Maurice B
Published: July 2021

Hypercoagulability increases the risk of thrombi that cause cardiovascular events. Dehydration and hypernatremia are often accompanied by thrombosis, but the mechanisms are not clear. Von Willebrand Factor is secreted by endothelium, affecting aggregation of platelets and promoting activation of the coagulation cascade and formation of thrombi. Here we show that in culture of primary Human Umbilical Vein Endothelia Cells, elevating medium osmolality to 320-380 mosmol/kg by adding NaCl reversibly increases both vWF mRNA and vWF secretion. The high NaCl increases expression of tonicity regulated transcription factor NFAT5 and its binding to promoter of vWF gene, suggesting that vWF upregulation is caused by hypertonic signaling. To elevate NaCl in vivo, we modeled mild dehydration, subjecting mice to water restriction (WR) for 9 days by feeding them with gel food containing 30% of water. Such WR elevates blood sodium from 145.1±0.5 to 150.2±1.3 mmol/l and activates hypertonic signaling as evidenced from increased expression of NFAT5 in tissues. WR increased vWF mRNA in liver and lung and raised vWF protein in blood. Immunostaining of liver revealed increased production of vWF protein by endothelium and increased number of microthrombi inside capillaries. WR also increased blood level of D-dimer, a fibrinogen degradation product indicative of ongoing coagulation and thrombolysis. We conclude that elevation of extracellular sodium within the physiological range raises expression and secretion of von Willebrand Factor sufficiently to increase coagulability of blood and risk of thrombosis. The results suggest that hydration and salt intake are modifiable factors that affect coagulability and thrombosis through high salt-dependent secretion of vWF from endothelial cells.
Abstract 374: Inhibitor of Differentiation 3 Promotes High Fat--Induced Expansion of Committed Inflammatory Adipocyte Progenitor Cells
Authors: Kaplan Jennifer L , Harmon Daniel B , Marshall Melissa A , McNamara Coleen A
Published: July 2021

Background:
Global deletion of helix-loop-helix (HLH) transcription factor Inhibitor of Differentiation 3 (Id3) attenuates high fat diet (HFD) induced obesity and adipose tissue inflammation. Specifically,

Id3
-/-

mice have less epididymal adipose tissue (eAT), fewer proinflammatory macrophages in eAT after HFD, and attenuated HFD-induced levels of MCP-1 mRNA and protein in eAT. It is currently unclear how Id3 is regulating MCP-1 or macrophage accumulation in eAT.

Methods & Results:

Id3
-/-

mice and

Id3
+/+

littermates were fed 60% HFD (n=7, 5) or standard chow (n=6, 7) for 1 week. Intracellular levels of MCP-1 in stromavascular fraction (SVF) cells from eAT were analyzed via flow cytometry. HFD increased the number of MCP-1
hi
cells in

Id3
+/+

mice compared to chow controls (8.66 vs 2.36 x10
4
, p<0.05), but there was no increase in

Id3
-/-

mice (3.28 vs 1.53 x10
4
, n.s.). Phenotyping of the SVF by flow cytometry revealed that MCP-1
hi
cells were mostly adipocyte progenitor cells (AdPCs) (78.8±6.8%), recently characterized as CD31
-
CD45
-
Ter119
-
CD34
+
CD29
+
Sca-1
+
. Interestingly, HFD induced an Id3-dependent increase in AdPCs. It was recently shown that CD24
-
AdPCs are more committed to the adipocyte lineage than CD24
+
AdPCs. CD24
+
AdPCs lacked MCP-1 expression, but committed CD24
-
AdPCs expressed MCP-1. To determine proliferation of AdPCs, BrdU was i.p. injected during the 1 week HFD (

Id3
+/+

n=11,

Id3
-/-

n=7).

Id3
+/+

mice had a HFD-induced increase in proliferating AdPCs (11.71% vs 5.71%, p<0.01), while,

Id3
-/-

mice lacked an increase (7.96% vs 6.69%, n.s.). Finally, OP-9 and 3T3-L1 preadipocyte cell lines were transfected with a p21
Cip1
luciferase promoter construct. Co-transfection with Id3 reduced p21
Cip1
promoter activation by 2-fold (p<0.01).

Conclusion:
Committed AdPCs are the predominant source of MCP-1 in the early stages of high fat feeding, and diet-induced expansion of these AdPCs is dependent on Id3. Id3 inhibition of p21
Cip1
promoter activation in AdPCs may be one potential mechanism mediating this effect. This novel evidence of the inflammatory properties of AdPCs and the role that Id3 plays in their expansion during obesity may lead to new therapeutic targets for the treatment of diet-induced adipose inflammation and its metabolic consequences.
Abstract 382: Oxidized LDL Signaling “Primes” GPVI-Mediated Platelet Activation in a CD36-Dependent Manner
Authors: Kartz Gabriella , Yang Moua , Sanchez Andrea , Silverstein Roy
Published: July 2021

CD36 acts as an important participant in the prothrombotic state associated with chronic inflammatory diseases such as atherosclerosis and diabetes by serving as a signaling relay point for danger-associated molecular patterns (DAMPs), including oxidized low-density lipoproteins (oxLDL) and advanced glycated proteins. Although CD36-mediated signaling pathways have been well characterized in macrophages, less is known about CD36-mediated signaling in platelets. Our group identified important roles for specific Src family kinases (SFK) Lyn and Fyn, and for the guanine nucleotide exchange factors Vav1 and Vav3. Since platelet activation by the collagen receptor GPVI also involves downstream activation of SFKs, we now hypothesize that oxLDL signaling via CD36 primes platelets for hyperactivity by activating components of the GPVI signaling pathway. To test this hypothesis, we treated fluorescently labeled murine platelets with either native LDL (nLDL) or oxLDL and exposed them to immobilized collagen under defined shear flow in a microfluidic flow chamber. A significant increase (~1.2 fold) in platelet accumulation was observed with oxLDL treatment. No increase in accumulation was observed in oxLDL-treated platelets from cd36 null mice, suggesting that this phenomenon was CD36-dependent. Furthermore, addition of tirofiban ablated the oxLDL-induced increase in platelet accumulation, suggesting that the increase was likely due to enhanced platelet-platelet contacts (i.e. enhanced platelet activation). This supports previous data from our laboratory showing that treatment of murine platelets with oxLDL alone resulted in activation of αIIbβ3 integrin. Additionally, oxLDL-treated platelets from vav1/vav3 double null mice phenocopied cd36 null platelets and displayed no increase in platelet accumulation compared to nLDL-treated platelets, strengthening the functional link between CD36 and GPVI signaling pathways. These data suggest a functional link between CD36 and GPVI signaling pathways in platelets, which may contribute to platelet hyperactivity in athero-inflammatory diseases.
Abstract 41: SH2B3 Is a Genetic Determinant of Cardiac Inflammation and Fibrosis
Authors: Flister Michael J , Hoffman Matthew , Lemke Angela , Prisco Sasha , Rudemiller Nathan , O'Meara Caitlin , Moreno Carol , Geurts Aron , Lazar Jozef , Adhikari Neeta , Hall Jennifer , Jacob Howard
Published: July 2021

Background:
Genome wide association studies (GWAS) are powerful tools for nominating pathogenic variants, but offer little insight as to how candidate genes impact disease outcome. Such is the case for SH2B adaptor protein 3 (SH2B3), which is associated with coronary artery disease (CAD), atherosclerosis, and risk of myocardial infarction (MI), but its role in post-MI response is completely unknown.

Methods:
Using an experimental model of MI (left anterior descending artery [LAD] occlusion) in wild-type (WT) and Sh2b3 knockout (KO) rats, we assessed the role of Sh2b3 in post-MI fibrosis, leukocyte infiltration, angiogenesis, left ventricle (LV) contractility, and inflammatory gene expression. We also confirmed our findings in LV samples from end-stage heart failure patients with or without the MI-associated SH2B3 risk allele.

Results:
Compared with WT, Sh2b3 KO rats had significantly increased fibrosis (2.2-fold; P2-fold; P<0.001), which coincided with decreased LV fractional shortening (FS) (-Δ11%; P<0.05) at 7 days post-LAD occlusion. Despite an increased angiogenic potential in Sh2b3 KO rats (1.7-fold; P<0.05), we observed no significant differences in LV capillary density between WT and Sh2b3 KO rats. Of the 903 genes examined, 19 were significantly elevated in the post-LAD occluded hearts of Sh2b3 KO rats relative to WT, of which three (NLRP12, CCR2, and IFNγ) were also significantly elevated in the LV of heart failure (HF) patients carrying the MI-associated rs3184504 [T] SH2B3 risk allele.

Conclusions:
These data suggest for the first time that SH2B3 is a master risk factor for MI by impacting both MI incidence and post-MI response.
Abstract 412: The Sodium-Glucose Cotransporter 2 Inhibitor Empagliflozin Improves Diabetic Complications in the Streptozotocin Type 1 Diabetes Mellitus Model by Interfering With Glucotoxicity and Rescue of Beta-Cell Function
Authors: Welschof Philipp , Oelze Matthias , Kröller-Schön Swenja , Jansen Thomas , Hausding Michael , Mikhed Yuliya , Stamm Paul , Mader Michael , Zinßius Elena , Agdauletova Saule , Wenzel Philip , Schulz Eberhard , Bottari Serge , Mayoux Eric , Münzel Thomas , Daiber Andreas
Published: July 2021

Objectives:
In diabetes, cardiovascular complications are associated with endothelial dysfunction and oxidative stress. Empagliflozin (Empa), as a selective sodium-glucose co-transporter 2 inhibitor (SGLT2i) in clinical development, offers a promising novel approach for the treatment of type 2 diabetes by enhancing urinary glucose excretion. The aim of the present study was to test whether treatment with Empa could improve endothelial dysfunction in type I diabetic rats via reduction of glucotoxicity and associated oxidative stress.

Research Design and Methods:
Type I diabetes in Wistar rats was induced by an intravenous injection of streptozotocin (60 mg/kg). One week after injection Empa was administered via drinking water for 7 weeks.

Results:
Treatment with Empa (10 and 30 mg/kg/d), showed reduction of blood glucose and a normalization of endothelial dysfunction (aortic rings) in diabetic rats and a reduced oxidative stress in aortic vessels (dihydroethidine staining) and in blood (phorbol ester/zymosan A-stimulated chemiluminescence). Additionally, the pro-inflammatory phenotype and glucotoxicity in diabetic animals was normalized by SGLT2i therapy.

Conclusion:
In this study we could demonstrate that Empa improves hyperglycemia and prevents the development of endothelial dysfunction and oxidative stress in type 1 diabetic rats. Future studies will investigate the underlying mechanisms of these antioxidant and anti-inflammatory effects with special emphasis on low-grade inflammation, glucotoxicity and oxidative stress, all of which contributes to cardiovascular complications.
Abstract 425: Development of a Vascular Cell Adhesion Molecule-1 Targeted Esterase Nanofiber that Binds Activated Endothelial Cells
Authors: Albaghdadi Mazen S , Rupert Perez Charles , Worthy Amanda , Bahnson Edward S , Carpenter Clifford , Muller William , Stupp Samuel S , Kibbe Melina R
Published: July 2021

Introduction:
Novel therapies capable of directly targeting and regressing atherosclerotic plaques would provide tremendous benefit to patients with atherosclerotic cardiovascular disease. Molecular imaging studies have shown the feasibility of targeting vascular adhesion molecule (VCAM-1) for non-invasive atherosclerosis imaging. Histidine contains an imidazole ring with esterase activity that can convert cholesterol esters to free cholesterol. Thus, we hypothesize that a peptide amphiphile (PA) functionalized with a VCAM-1 binding peptide and histidine will spontaneously form a targeted nanofiber that binds activated endothelium and liberates free cholesterol from cholesterol esters.

Methods:
PAs were synthesized by solid phase methods and purified by reversed phase HPLC. Three different VCAM-1 binding peptide sequences were conjugated to the PAs (free N-terminus, free C-terminus, and cyclic). To assess 3-dimensional structure of the co-assembled PAs, cryogenic transmission electron microscopy (TEM) was used. To assess targeting of the VCAM-PAs (50μM), human umbilical endothelial cells (HUVECs) were stimulated with TNFα (10ng/ml). To assess the ability of the histidine-PA to liberate free cholesterol, a modified Amplex Red Cholesterol Assay was used.

Results:
Using TEM, we demonstrated that the co-assembled VCAM- and histidine-PAs formed characteristic nanofibers with diameters of 5-8nm and lengths ranging from 300nm to 1μm. TNFα-activated HUVECs were confirmed to express VCAM-1 protein by Western blot analysis. Confocal imaging revealed that the VCAM-1 binding peptide with a free N-terminus conjugated to the PA had the greatest binding affinity to the TNFα-activated HUVECs. Co-incubation of the histidine-PA with cholesterol esters demonstrated that free cholesterol was liberated from cholesterol esters in a concentration-dependent manner with a five-fold increase in free cholesterol release with 25μM of PA versus 1μM.

Conclusion:
We have designed and characterized a VCAM-1 targeted nanofiber that successfully binds activated endothelium and generates free cholesterol from cholesterol esters. This novel therapeutic nanotechnology has the capacity to target and regress atherosclerotic plaques.
Abstract 43: The Hypofunctional GPER P16L Variant is Associated With a Gene Dosage-Related Increase in Plasma LDL Cholesterol
Authors: Hussain Yasin , Feldman Ross D , Ding Qingming , Chorazyczewski Jozef , Ban Matthew R , McIntyre Adam D , Gros Rob , Hegele Robert A
Published: July 2021

Introduction:
Estrogen deficiency is linked with dyslipidemia, especially in postmenopausal women, through a poorly understood mechanism. GPER is a recently recognized GPCR which is activated by estrogens. However, the role of GPER in mediating estrogen’s effects on lipid metabolism is unknown. We recently identified a common hypofunctional missense variant of GPER, namely P16L (allele frequency ~ 20%). We studied association of this with plasma LDL cholesterol levels. Further, we studied the role of GPER in regulating expression of the LDL receptor.

Methods:
Our discovery cohort was a genetically isolated population of Northern European descent (n=415), and our validation cohort consisted of 505 normal, healthy subjects 18-56 years of age from London, Ontario. Genomic DNA was extracted from whole blood and genotyped for GPER using a dedicated TaqMan assay. Additionally we examined the role of GPER on the regulation of LDL receptor expression by treatment with the GPER agonist, G1.

Results:
In the discovery cohort, the GPER P16L genetic variant was associated with a significant gene-dosage related increase in LDL cholesterol (CC [homozygous wild type] =3.18±0.84 (mean+SD); CT [heterozygote] =3.25±0.80; and TT [homozygous variant] =4.25±0.87 mmol/L, p<0.05). Total cholesterol concentrations followed a similar gradient across genotypes. In the validation cohort, the GPER P16L genetic variant was associated with a similar significant gene-dosage related increase in LDL cholesterol (CC =2.16±0.67; CT [heterozygote] =2.29±0.67; TT =2.40±0.84 mmol/L, p<0.05). In HepG2 cells expressing GPER, G1 mediated a concentration-dependent increase in LDL receptor expression. Pre-treating the cells with the GPER antagonist G15 attenuated the effect of G1 on LDL receptor upregulation. Further, downregulation of GPER expression via infection with a shGPER construct also attenuated G1's effect on LDL receptor upregulation.

Conclusion:
GPER activation upregulates LDL receptor expression. Further, carrying the hypofunctional P16L genetic variant of GPER, increases plasma LDL cholesterol in humans. In aggregate these data suggest an important role of GPER in regulation of LDL receptor expression and consequently LDL metabolism.
Abstract 434: LDL Apheresis Significantly Alters HDL Cargo and Function in Familial Hypercholesterolemia Subjects
Authors: Lynch Donald R , Wiese Carrie , Liu Qi , McDonald W H , Taylor Robert C , Landstreet Stuart R , Fazio Sergio , Vickers Kasey , Linton MacRae F
Published: July 2021

In addition to cholesterol, HDL transports a wide-variety of cargo including vitamins, nucleic acids, and a diverse set of proteins. Outside of the reverse cholesterol transport pathway, HDL has alternative functions that contribute to its anti-atherogenicity, including anti-inflammatory, anti-oxidant, and signaling capacities. Recently, we found that HDL transports and delivers functional microRNAs to recipient cells, which likely confers HDL’s ability to suppress adhesion molecule expression in endothelial cells. As such, changes to HDL’s cargo impact many of these alternative functions, and thus its protective capacity. HDL dysfunction has been identified among patients with familial hypercholesterolemia (FH), an inherited disease due to mutations in the LDL receptor and associated with severe elevations in LDL-cholesterol (LDL-C) levels, which may necessitate LDL apheresis (LA) in management. Here we demonstrate that LA significantly alters HDL’s miRNA and protein signatures. We believe that these changes may have profound consequences on HDL’s protective capacity. Using density-gradient ultracentrifugation, we found 31 proteins to be significantly altered on HDL after LA, as determined by shotgun proteomics and multidimensional protein identification technology analysis. For example, vitamin D-binding protein (1.74-fold) was increased, while lipopolysaccharide-binding protein (-1.92-fold), platelet-activating factor acetylhydrolase (-2.06), and apolipoprotein A-V (-2.4-fold) were found to be decreased. Gene ontology and KEGG enrichment analysis demonstrated roles of these proteins in response to stress, coagulation, hemostasis, and vesicle-mediated transport. Fast-protein liquid chromatography was used to further purify HDL for miRNA profiling using TaqMan OpenArrays, and we found 8 HDL-miRNAs to be significantly altered -- 3 down (miR-302b -13.9-fold, miR-224 -1.8, miR-572 -2.4-fold) and 5 up (miR-7 9.8-fold, miR-208b 16.2-fold, miR-34a-3p 3-fold, miR-627 2.4-fold, miR-1183 4.8-fold). Both miR-302b and miR-224 decreased; these are miRNAs previously reported to suppress proliferation through targeting AKT2 and apoptosis, respectively. As such, these changes likely alter HDL’s function in FH subjects.
Abstract 437: Lipoprotein(a) Internalization in HepG2 Cells is Regulated by Proprotein Convertase Subtilisin Kexin Type 9 Through the Low-Density Lipoprotein Receptor
Authors: Romagnuolo Rocco , Seidah Nabil G , Koschinsky Marlys L
Published: July 2021

Elevated levels of lipoprotein(a) (Lp(a)) have been identified as an independent and causal risk factor for coronary heart disease. Lp(a) consists of an LDL-like moiety covalently linked to the unique glycoprotein apolipoprotein(a) (apo(a)). The mechanism by which Lp(a) is catabolized is currently unknown, but may form the basis for the development of drug therapy to reduce high levels of plasma Lp(a). Although the role of the LDL receptor (LDLR) in Lp(a) catabolism is controversial, recent evidence has shown that Lp(a) levels are significantly reduced with an antibody against proprotein convertase subtilisin kexin type 9 (PCSK9) in patients with hypercholesterolemia receiving statin therapy. Therefore, we explored the role of PCSK9 in Lp(a)/apo(a) internalization by hepatic cells. Lp(a) or apo(a) internalization is significantly reduced in HepG2 (human hepatoma) cells either by overexpressing PCSK9 or by treatment with purified PCSK9. The ability of Lp(a) and apo(a) to be internalized was significantly reduced in the presence of the lysine analogue, ε-ACA, indicating lysine-dependent interactions with cellular receptors. Mutation of the strong lysine binding site in a recombinant apo(a) variant resulted in a reduced ability to be internalized. While LDL can bind to PCSK9 and inhibit its ability to degrade the LDLR, we found that Lp(a) lacked these properties. Interestingly, overexpressing the LDLR on HepG2 cells significantly increased the ability of Lp(a) to be internalized, an effect that was partially reduced by the addition of PCSK9. This indicates a potential key role for the LDLR in regulating Lp(a) catabolism. Furthermore, knockdown of clathrin heavy chain resulted in a significant decrease in apo(a) internalization and apo(a) internalization was not further reduced by pre-treatment of PCSK9 in the context of clathrin heavy chain knockdown. Treatment of HepG2 cells with a lysosomal inhibitor, but not a proteosomal inhibitor, resulted in accumulation of Lp(a) in HepG2 cells indicating that Lp(a) is potentially targeted for degradation through lysosomes. Taken together, these results indicate that Lp(a)/apo(a) uptake can be regulated in HepG2 cells by PCSK9 and the LDLR through clathrin-mediated endocytosis and lysosomal degradation.
Abstract 438: Peripheral but Not Central Glucagon-Like Peptide-1 Receptor Agonism Regulates Fasting Dyslipidemia by Reducing VLDL Production
Authors: Taher Jennifer , Baker Chris , Cuizon Carmelle , Naples Mark , Farr Sarah , Adeli Khosrow
Published: July 2021

Glucagon-like peptide-1 (GLP-1) is an incretin hormone that reduces plasma glucose levels and thus acts as an important drug target in the treatment of type 2 diabetes (T2D). Our laboratory has further shown that GLP-1 regulates intestinal lipoprotein metabolism and may also play a similar role in the liver. Hepatic lipids are packaged with apolipoprotein B-100 (apoB100) into very low density lipoproteins (VLDL) and secreted into the plasma. Dysregulation of VLDL production results in the fasting dyslipidemia that is observed in T2D. We postulate that GLP-1 receptor (GLP-1R) agonism regulates fasting dyslipidemia by decreasing VLDL production in insulin resistance through a peripheral or central pathway. To test this, experiments were conducted in fructose-fed insulin resistant Syrian golden hamsters. Hamsters were given twice daily intraperitoneal(i.p) injections of the GLP-1R agonist exendin-4 (ex4; 5nM/kg) for 7 days and placed into metabolic cages. Plasma was collected for lipid analysis following i.p poloxamer to prevent lipoprotein uptake and livers were excised. Peripheral ex4 prevented fructose-induced fasting dyslipidemia and decreased fasting plasma- and VLDL-triglyceride (TG), -cholesterol and -apoB100 accumulation. Ex4 also reduced food intake and body weight and thus pair-fed controls were added. Pair-feeding did not account for the full ex4 lipid-lowering effect. In comparison to both controls, ex4 treated hamsters had reduced respiratory exchange ratio and CO
2
production indicating a switch from glucose to fat metabolism as the main source of energy, and also decreased hepatic mRNA markers for
de novo
lipogenesis. To determine involvement of a central pathway, hamsters received a 4 day intracerebroventricular administration of ex4 (250ng) into the third ventricle. Central ex4 reduced body weight and food intake but did not modulate fasting plasma and VLDL-lipid or apoB100 accumulation, further suggesting that decreases in food intake cannot solely explain the changes in VLDL production. Our studies suggest that GLP-1R agonism reduces fasting dyslipidemia in insulin resistance by decreasing VLDL production through a peripheral pathway. GLP-1R agonism may be a potential therapy in the treatment of fasting dyslipidemia.
Abstract 439: REV-ERB--Mediated Regulation of Cholesterol Biosynthesis and Atherosclerosis
Authors: Sitaula Sadichha , Solt Laura , Kamenecka Theodore , Burris Thomas P
Published: July 2021

Despite significant research efforts in the field of cardiovascular diseases, atherosclerotic cardiovascular disease remains the leading cause of mortality. Current treatment options available to lower lipids are less than optimal in their ability to alter the progression of atherosclerosis. Therapies that can target both metabolic and inflammatory aspect of this disease have significant potential in the treatment of cardiovascular diseases. Recent work from our lab and others suggests that the nuclear receptor REV-ERB plays important role in regulating both lipid levels and inflammatory processes. We hypothesize that synthetic REV-ERB ligands can be used as tools to investigate the biology of the REV-ERBs in the regulation of cholesterol biosynthesis and that these ligands have therapeutic potential in the treatment of atherosclerosis. Eight week old mice (n=11 per group) with deficient LDL receptor were fed high cholesterol diet and treated with vehicle (15% cremophor) or REV-ERB agonist, SR9009 (100mg/kg, I.P.) for a period of eight weeks. We demonstrate that treatment of ldlr-deficient mice with SR9009 significantly reduces the expression of genes involved in cholesterol synthesis pathway in liver and considerably lowers plasma lipid levels - 45% reduction in LDL and 25% reduction in total cholesterol levels. In differentiating macrophages treated with SR9009, significant changes were observed in expression of genes involved in inflammatory responses. These results suggest that REV-ERBs have important role in the regulation of cholesterol synthesis and macrophage biology with therapeutic potential in the treatment of metabolic disorders, including atherosclerosis.
Abstract 442: D-4F, an Apolipoprotein A-I Mimetic Peptide, Protects Macrophages From Oxidized Low-Density Lipoprotein--Induced Apoptosis by Inhibiting the Endoplasmic Reticulum Stress-C/EBP Homologous Protein Pathway
Authors: Yao Shutong , Tian Hua , Miao Cheng , Zhao Li , Jiao Peng , Yang Nana , Sang Hui , Yu Yang , Wang Xinnong , Qin Shucun
Published: July 2021

Objective:
D-4F, an apolipoprotein A-I (apoA-I) mimetic peptide, exerts a variety of atheroprotective functions similar to apoA-I, the major protein component of high density lipoprotein (HDL), including acting as an antioxidant, mediating cholesterol efflux from foam cells and direct anti-inflammatory effects. Our previous studies have demonstrated that endoplasmic reticulum (ER) stress promotes macrophage-derived foam cell formation by upregulating CD36 expression and mediates oxidized low-density lipoprotein (ox-LDL)-induced macrophage apoptosis. The goal of this study was to investigate the protective effect of D-4F on ox-LDL-induced macrophage cytotoxicity and specifically the ER stress-C/EBP homologous protein (CHOP) pathway-mediated apoptosis.

Methods and Results:
Treatment with D-4F (12.5, 25 and 50 mg/L) attenuated ox-LDL (100 mg/L)-induced cholesterol accumulation in RAW264.7 macrophages and foam cell formation in a dose-dependent manner. Similar to tunicamycin (TM), a classical ER stress inducer, ox-LDL reduced cell viability and induced apoptosis in RAW264.7 macrophages. The cytotoxic effects of ox-LDL (100 mg/L) and TM (5 mg/L) were remarkably inhibited by D-4F treatment. Interestingly, we found that D-4F also significantly suppressed the ox-LDL- and TM-induced CD36 upregulation and activation of ER stress signaling events, including the phosphorylation of inositol-requiring enzyme 1 (IRE1) and nuclear translocation of activating transcription factor 6 (ATF6). In addition, exposure of RAW264.7 macrophages to ox-LDL or TM resulted in a significant increase in the expression of CHOP, a proapoptotic transcription factor regulated by IRE1 and ATF6 under conditions of ER stress. D-4F blocked these effects in a dose-dependent manner. Moreover, administration of apoE
–/–
mice with D-4F (1 mg/kg per day) suppressed apoptosis and the upregulation of CD36, phospho-IRE1, GRP78 and CHOP in macrophage-dense atherosclerotic lesions.

Conclusion:
These data indicate that D-4F can protect macrophages from ox-LDL-induced apoptosis and that the mechanism at least partially involves its ability to inhibit the ER stress-CHOP signaling pathway.
Abstract 485: Effect of Bone Marrow Transplantation on Aortic Inflammatory Disease and Accelerated Atherosclerosis
Authors: Douglas Gillian , Patel Jyoti , Hale Ashley , Channon Keith M
Published: July 2021

In ApoE-/- mice Ang II infusion leads to vascular inflammation resulting in the formation of aortic aneurysms and accelerated atherosclerosis. In order to elucidate the role of hemopoetic cells in these pathologies, bone marrow transplant (BMT) has been utilized. However, if BMT is used, it is critical that we fully understand the effect of BMT on aortic inflammatory disease and atherosclerosis. In this study we used Ang II to investigate the effects of bone marrow transplant on aortic inflammatory disease and accelerated atherosclerosis.

ApoE-/- mice (12wk age) were either lethally irradiated (2x 5Gy) and reconstituted with ApoE-/- bone marrow (1x10-6 cells) or non-irradiated. . BMT mice and non-irradiated littermates were treated with Ang II (0.8mg/kg/day) to induce aortic inflammation and accelerate atherosclerosis. After 14 days of treatment mice were culled and the aorta and aortic root harvested. AngII infusion in non-irradiate mice resulted in a 33% incidence in aortic aneurysm, however, in BMT mice no aneurysms were observed. Ang II infusion lead to an increase in atherosclerosis with a significant increase in inflammatory cell infiltration observed. Irradiation resulted in a significant decrease in Ang II-induced accelerated atherosclerosis (0.049±0.003 vs. 0.037±0.016; P<0.05; non-irradiated vs. irradiated) and a significant reduction in macrophage infiltration (0.018±0.003 vs. 0.007±0.002; P<0.05; non-irradiated vs. irradiated). However, in order to achieve a similar incidence of aortic aneurysms in BMT mice as observed in non-BMT mice treated with 0.8 mg/kg/day Ang II, the dose of Ang II had to be increased to 3mg/kg/day. A similar result was observed in atherosclerosis studies, with a dose of 3mg/kg/day required to induce comparable levels of macrophage infiltration and plaque size to those in non-BMT mice treated with a dose of 0.8mg/kg/day.

In conclusion, BMT resulted in a significant decrease in AngII-induced accelerated atherosclerosis and a reduction in Ang II-mediated aortic inflammation. Interestingly, BMT mice had a significant reduction in inflammatory cell infiltration compared with non-irradiated littermates indicating that the reduction maybe due to an alteration in inflammatory cell recruitment after BMT.
Abstract 497: Carotid Stiffness and Atheromasic Plaque Composition: Biomolecular Assessment
Authors: Bitto Alessandra , Galfo Federica , Zito Concetta , De Caridi Giovanni , Squadrito Francesco
Published: July 2021

The different histopathology of atherosclerotic plaques may modify carotid stiffness. In this work we evaluated if a correlation exists between stiffness, type of plaques, inflammatory and apoptotic molecules and Osteoprotegerin (OPG) expression. 19 patients were divided in two groups depending on type of plaque (Mixed and Fibrocalcific) established through Doppler-ultrasound. Among inflammatory molecules we considered p-JNK ,p-ERK and NLRP3 levels by Western Blotting analysis whereas among apoptotic molecules we evaluated TNFα and TNFαIIR, FAS and FAS-L, Bcl-2, Casp8, ASK1, OPG and HMGB1 levels by Real Time PCR. We observed increased OPG protein levels in mixed plaques compared with fibrocalcific ones according to expectations, because those plaques start to calcificate, as well as increased levels of all markers of inflammation and apoptosis, confirming a predictive role of these molecules relative to plaque composition. Stiffness and the Augmentation Index does not result affected to plaque composition but may depend on reduced size of patients recruited.
Abstract 5: ApoAI Binds to Phosphatidylinositol 4,5 Bisphosphate (PIP2), Which is Exposed on the Cell Surface by Novel PIP2 Floppase Activity of ABCA1, and Promotes Cholesterol Efflux
Authors: Gulshan Kailash , Brubaker Gregory , Hazen Stanley , Smith Jonathan
Published: July 2021

Introduction:
ApoAI-ABCA1 mediated nascent HDL assembly promotes cholesterol efflux and reverse cholesterol transport, but the mechanism of HDL assembly is unknown.

Objective:
To determine role of PIP2 in ApoAI-ABCA1 mediated nascent HDL assembly.

Methods and Results:
To determine which lipid species are directly bound by apoAI, a lipid-protein overlay assay was performed using PIPstrips and sphingostrips from Echelon Biosciences. ApoAI bound directly to phosphatidylinositol phosphates (PIPs) rather than PC, PS, PE, SM or cholesterol. This interaction was not solely electrostatic, as PIP3, the most negative charged PIP had less apoAI binding than various PIP2 species. ApoAI did not bind to other charged lipids such as sphingosine 1-P, PI(3)-P, PI(4)-P, and PI(5)-P. Surface plasmon resonance assays using immobilized apoAI or PIP2 were performed, showing that apoAI interacted reversibly with PI(4,5)P2 in a dose dependent manner at the nM concentration range. The central domain of apoAI (residues 44-185) was sufficient for PIP2 binding. In RAW264.7 macrophages, depletion of cellular PIPs by treatments with PI3K inhibitor or PTEN inhibitor decreased cholesterol efflux by 45±1.4% and 41±6.4% respectively (p<0.005). Degradation of cell surface PIP2 via treatment with PI specific PLC decreased cell surface binding of apoAI by 37±6% and cholesterol efflux by 48±8% (p<0.005). In mouse bone-marrow derived macrophages, cholesterol efflux to apoAI was increased ~47% by GRIP (PIP2 binding PH domain of PLCδ) and decreased 41±8% by PTEN inhibition (p<0.005). Liposomes made with POPC:POPS:FC:PIP2 (65:20:10:5 mole ratio) were solubilized by apoAI at 37
o
C, pH5, but apoAI had little activity using similar liposomes without PIP2 . ABCA1 expression led to 2-fold higher cell surface PIP2, assayed by flow cytometry using a PIP2 specific antibody. RAW cells stably transfected with PIP2 binding domain 2X PH-PLCδ-eGFP showed enrichment of this PIP2 reporter on the plasma membrane, while expression of ABCA1 led to partial redistribution to cytoplasm, consistent with ABCA1 having PIP2 floppase activity.

Conclusions:
ApoAI has novel PIP2 binding activity, ABCA1 is a novel PIP2 floppase, and cell surface PIP2 is required for optimal cholesterol acceptor activity of apoAI.
Abstract 50: High-Density Lipoprotein Protects Macrophages Against Apoptosis in a PDZK1-Dependent Manner
Authors: Yu Pei , Zhang Yi , Trigatti Bernardo
Published: July 2021

Macrophage apoptosis contributes to the formation of necrotic cores in atherosclerotic plaques, increasing the risk of plaque rupture, leading to clinical heart attack. Previous work from our group showed that HDL is able to protect macrophages against apoptosis, and that such protection requires HDL’s receptor protein scavenger receptor class B, type I (SR-BI). In this study, we hypothesize that this protection requires not only SR-BI, but also SR-BI’s adaptor protein PDZK1. We incubated mouse peritoneal macrophages with or without lipoproteins and triggered apoptosis with thapsigargin or tunicamycin. Cell apoptosis was assessed by annexinV, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and cleaved caspase-3 staining. Only low levels (~5% of cells) of apoptosis were observed in macrophages treated with thapsigargin or tunicamycin when they were cultured with lipoproteins. However thapsigargin or tunicamycin stimulated apoptosis increased ~4 fold when lipoproteins were removed from the culture medium. The addition of 50μg/ml of purified HDL (P<0.05), but not LDL, to lipoprotein-deficient medium suppressed apoptosis. However, HDL did not inhibit cell apoptosis when PDZK1 was deleted. Our time-course study showed that incubation with HDL increased phosphorylation of Akt1, Foxo3a and Erk1/2 and reduced Bim in wild type (WT), but not PDZK1-/- macrophages. In order to test the role of PDZK1 in vivo, LDLR-/- mice were transplanted with either PDZK1-/- (n=12) or WT (n=12) bone marrow and challenged with high fat diet to induce atherosclerosis. The cross-sections of aortic sinus were stained with H&E to visualize plaques. The relative necrotic core area was significantly larger (P<0.05) in PDZK1-/- group (98187.2±8076.1) than that in WT control (27279.6±16688.7). Also, compared to LDLR-/- mice transplanted with WT bone marrow, the ones with PDZK1-/- bone marrow have 2.2-fold increased TUNEL(+) (P<0.05) and 3.6-fold increased cleaved caspase-3(+) (P<0.01) macrophages in plaques. In conclusion, PDZK1 is required for the HDL-dependent protection against macrophage apoptosis. In atherosclerotic plaques, PDZK1 plays a role in protecting macrophages from apoptosis and suppressing the development of necrotic cores.
Abstract 512: Collagen Homologous Sequence R1R2 Mediates Vascular Remodeling by Decreasing Inflammation and Smooth Muscle Proliferation
Authors: Lee Ting-Hein , Chiang Hou-Yu
Published: July 2021

The extracellular matrix (ECM) is a major constituent of the vessel wall. Except for providing a structural scaffold for cells, ECM controls numerous cellular functions like adhesion, growth, migration and differentiation. The components of ECM are mediated by the interplay between ECM synthesis, deposition, degradation and the interaction between ECM proteins. Vascular remodeling occurs in the vascular diseases and is characterized by endothelial cell activation, inflammatory cell infiltration, smooth muscle cell (SMC) proliferation/migration, and augmented deposition of ECM proteins. Collagen I is the major ECM component in the arterial wall, excess collagen I accumulation may exacerbate the vascular disease by further facilitating SMC proliferation/migration. Therefore, treatments to inhibit excess collagen deposition could provide a remedy for vascular disease. R1R2, a peptide derived from the bacterial adhesin SFS with sequence homology to collagen, is known to inhibit collagen I deposition by inhibiting the binding of fibronectin to collagen. Studies have revealed that R1R2 affects collagen I-dependent cell growth and migration in vitro. However, the in vivo functions of R1R2 during vascular remodeling remain unknown. We hypothesized that R1R2 prevents excess collagen I accumulation and SMC proliferation, resulting in decreased neointimal formation. We induced vascular remodeling by ligating the carotid artery on mice. Delivery of R1R2 was periadventially applied using pluronic gel and evaluated its effects on vascular remodeling, ECM deposition, SMC proliferation and differentiation. Morphometric analysis demonstrated that R1R2 reduced intima-media thickening by 50% compared to the control group. R1R2 treatment also decreased collagen I deposition in the vessel wall and maintained SMC in the contractile phenotype. Interestingly, R1R2 dramatically reduced inflammatory cell infiltration into the vessel by 80% accompanied with decreased VCAM-1 and ICAM-1. In conclusion, our data showed that R1R2 attenuates the vascular remodeling response by decreasing inflammation and SMC proliferation/migration. These studies provide a therapeutic potential of periadventitially delivering R1R2 in treating vascular diseases.
Abstract 513: Endothelial Barrier Function is Maintained by the RNA-Binding Protein Quaking
Authors: de Bruin Ruben G , Dane Martijn J , Lee DaeHyun , van der Veer Eric P , Roeten Marko K , Schmidt Iris , Sundararajah Janani , Bijkerk Roel , de Boer Hetty C , Rabelink Ton J , van Zonneveld Anton-Jan , van Gils Janine M
Published: July 2021

Endothelial barrier function plays a major role in the onset of atherosclerosis. This barrier is maintained largely by adherens junctions. Remarkably, little is known about their regulation at the post–transcriptional level. We found that the RNA-binding protein Quaking (QKI), known for its function in embryonic blood vessel formation, is highly expressed in quiescent endothelial cells (EC) in vivo. In vitro, EC displayed increased levels of QKI when cultured under laminar, atheroprotective flow. Using KLF2 overexpression and a human QKI promoter reporter gene, we found that KLF2 mediates this increase in QKI expression.

Subsequently, we aimed to investigate the role of QKI in EC vascular integrity. Interestingly, the mRNA of VE-cadherin, the prime adhesion protein in EC adherens junctions, contains a conserved QKI-binding site. We identified that the targeted reduction of QKI results in a reduction of VE-cadherin expression and organization at the cell periphery. These studies revealed a direct role for QKI in regulating VE-cadherin mRNA biology, as RNA-immunoprecipitation and luciferase-reporter assays revealed that QKI can directly bind to the VE-cadherin mRNA and induce transcript translation (4 fold ± 0.4; p<0.01), respectively. This effect was perturbed when the QKI-binding site was mutated. These results suggest that QKI acts to enhance barrier function. Overexpression of QKI markedly increased the capacity to form a high resistance endothelial monolayer (1.3 fold ± 0.96), while silencing of QKI markedly impaired EC barrier function (0.65 fold ± 0.13; p<0.05). To validate in vivo, we measured Bradykinin-induced vascular leakage in QKI viable mice (QKIv), which express decreased levels of the QKI protein. Indeed, QKIv mice displayed a 20% (p<0.05) increase in extravascular accumulation of Evans blue-labeled albumin compared to WT littermates.

In conclusion, we show that QKI functions as a critical regulator of VE-cadherin, and the modulation of QKI expression affects endothelial monolayer integrity. These studies provide novel insight into the importance of post-transcriptional regulation on endothelial barrier function, and may have wide ranging implications for the preservation of vascular integrity in disease.
Abstract 518: Differential Effects of Alcohol and Its Primary Metabolite Acetaldehyde on Vascular Smooth Muscle Cell Notch Signaling and Growth
Authors: Hatch Ekaterina , Morrow David , Hamm Katie , Cahill Paul A , Redmond Eileen M
Published: July 2021

Moderate consumption of alcohol (ethanol/EtOH) is a negative risk factor for atherosclerosis while binge drinking and chronic alcohol abuse is associated with increased cardiovascular disease and mortality. As the balance between the levels of EtOH itself and its primary metabolite, acetaldehyde, is likely different in these two drinking groups, we assessed the hypothesis that differential effects of EtOH and acetaldehyde on vascular smooth muscle cell (SMC) Notch signaling and growth may be key in mediating the effects of various drinking patterns on cardiovascular disease.

Human coronary artery smooth muscle cells (HCASMC) were treated (24 h) with either EtOH (25 mM) or acetaldehyde (10 or 25 μM). We confirmed our previously reported inhibitory effects of EtOH on Notch signaling and subsequently on the growth of HCASMC. In contrast, acetaldehyde treatment stimulated HCASMC proliferation (determined by cell counts) and increased proliferating cell nuclear antigen expression, concomitant with stimulation of Notch signaling as determined by increased Notch target gene (HRT 1-3) mRNA levels. Interaction of a ligand (e.g., Dll4) with the Notch receptor initiates proteolytic cleavage by γ-secretase, resulting in the release of the active Notch-intracellular domain (NICD). A fluorogenic peptide cleavage assay demonstrated almost complete inhibition by EtOH of Dll4-stimulated γ-secretase activity in a solubilized membrane fraction from HCASMC (similar to the effect of the control inhibitor, DAPT), but no effect of acetaldehyde treatment. Similarly, EtOH, but not acetaldehyde, affected the association and distribution of the γ-secretase catalytic subunit, presenilin-1, with lipid rafts as determined by dual fluorescent labeling and confocal microscopic visualization (Alexa 488-conjugated cholera toxin B subunit for lipid rafts, and Alexa 594-conjugated anti-presenilin-1 antibody).

In conclusion, EtOH and acetaldehyde have opposite effects on SMC Notch signaling and growth. These differential effects of alcohol and its metabolite may be relevant in mediating the opposing effects of different drinking patterns (e.g., daily moderate vs weekend binge) on cardiovascular disease.
Abstract 526: The Influence of Endothelium on Smooth Muscle-Derived α1-Integrin, ROS and Collagen IV Expression: Effects of High Glucose and Diabetes
Authors: Ledet Thomas , Smidt Kamile , Mumm Birgitte
Published: July 2021

The abnormal metabolism in diabetes facilitates the development of atherosclerotic cardiovascular disease. Consequently, can HUVECs (human-umbilical-vascular-endothelial-cells) transiently primed by high glucose (HG) or diabetic serum (DM) change HuVSMCs (Human-Vascular-Smooth- Muscle-Cells)-derived α1-integrin, ROS (Reactive Oxygen Species) and collagen IV expression? Is there a relationship between HuVSMCs-created α1-integrin expression, ROS production and collagen IV accumulation?

Conditioned medium (CM) from HUVECs, transiently primed by HG (CM
HG
) or DM (CM
DM
), was added to HuVSMCs. The expression of HuVSMCs-derived α1-integrin was measured with an in situ ELISA and the quantity of HuVSMCs-produced ROS was evaluated applying dihydro-rhodamine. The accumulation of HuVSMCs-secreted collagen IV was estimated in the medium by an ELISA. The isolated effect of reduced HuVSMCs-derived α1-integrin expression was elucidated by knock-down using siRNA whereas the impact of NADPH oxidase on HuVSMCs-derived ROS production was estimated using NADPH inhibition (Diphenyleneiodonium(DPI)).

CM
HG
and CM
DM
reduced HuVSMCs-derived α1-intergin expression while collagen IV and ROS production was increased. Immunopurified HSP90α from CM
HG
gave similar results whereas treatment with recombinant HSP90α has no impact. However, knock-down of α1-intergin increased HuVSMCs-derived collagen IV and ROS accumulation whereas reduced NADPH oxidase activity decreased ROS and collagen IV production but increased α1-intergin expression.

Previously, we showed that CM
HG
has increased pHSP90α (phosphorylated HSP90α) and now our observations suggest that HUVECs-derived pHSP90α down-regulate the expression of HuVSMCs-derived α1-integrin. The reduced presences of α1-integrin increased ROS and collagen IV expression from HuVSMCs. Consequently, HG and DM might via HUVECs-secreted pHSP90α act as potent modifiers of injury-induced changes within the arterial wall in diabetes.