bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 346: Manipulating P2Y2 Receptor Biased Signaling to Limit Pro-thrombotic Gene Expression in Human Coronary Artery Endothelial Cells
Authors: Zhang Lingxin , Wang Chuan , Alqurashi Thamer , Shen Jianzhong
Published: June 2021

Recently we reported that in human coronary artery endothelial cells (HCAEC), the P2Y2 nucleotide receptor (P2Y2R) regulates the pro-thrombotic gene tissue factor (TF) expression through positive and negative signaling mechanisms. Here, we report the identification of a P2Y2R ligand JZS0312 which selectively activates the negative, but not the positive pathways, leading to decreased TF expression. Unlike the endogenous agonist UTP, JZS0312 alone had no effect on TF mRNA transcription, but it unexpectedly inhibited UTP-induced TF expression. Mechanistic study showed that JZS0312 induced intracellular Ca
2+
mobilization in HCAEC which exhibited the same efficacy as UTP and was inhibited by AR-C118925, a P2Y2R-selective antagonist. However, when evaluated in their activation of the MAPK pathways, JZS0312 only activated the ERK1/2, but not JNK and p38, whereas UTP activated all the three, suggesting JZS0312 as a biased ligand for the P2Y2R. This notion was supported by the fact that JZS0312-induced ERK1/2 activation was inhibited by AR-C1189258 and it had no effect on ERK1/2 in P2Y2R-null cells. In addition, JZS0312 selectively activated the TF gene repressor Fra-1, but not the positive AP-1 subunits c-Jun and ATF-2, whereas UTP activated all of them. Furthermore, luciferase activity assay indicates that JZS0312 suppressed TF gene promoter activity via ERK1/2 activation, while UTP increased overall TF promoter activity. These findings reveal that JZS0312 acts as a biased agonist in human endothelial P2Y2R, in which it selectively activates the ERK1/2-Fra1 pathway, leading to suppression of TF gene transcription. Thus, JZS0312 represents the first identified biased ligand in the P2Y receptor family, opening new avenue for potential drug discovery in relevant inflammatory and thrombotic diseases.
Abstract 350: Synthetic Soluble MHC-I/peptide Complexes Confirm Activation of Functional Antigen-specific CD8+ T cells by Immunization With apoB-100-derived Peptide p210 in ApoE-/- Mice
Authors: Zhao Xiaoning , Dimayuga Paul C , Yano Juliana , Zhou Jianchang , Lio Wai Man , Cercek Bojan , Shah Prediman K , Chyu Kuang-Yuh
Published: June 2021

Background:
Investigations in our laboratory identified endogenous, antigen-specific CD8+ T cells reactive to apoB-100 related peptide in apoE-/- mice using fluorescent synthetic soluble MHC-I/peptide complexes called Pentamers (Pent). We hypothesized that immunization of apoE-/- mice with the apoB-100 peptide p210, which we and others have reported to reduce atherosclerosis, will result in the activation of a specific CD8+ T cell population that can be detected by Pent analysis.

Methods and Results:
Binding of the p210 peptide to the mouse MHC-I allele H2Kb was determined in a prior study. A p210-Pent library was generated to screen splenocytes from p210 immunized apoE-/- mice. Mice were immunized with p210 conjugated to cBSA with Alum (p210) at 7, 10 and 12 weeks of age then euthanized at 13 weeks of age. PBS and cBSA/Alum (cBSA) treatment served as controls. The screening assay identified 2 potential Pents that could discriminate the immunized mice from the controls. The Pent with the largest difference, called Pent 5, was selected for further study. Pent 5(+)CD8+ T cells in p210 immunized mice were significantly increased compared to PBS and cBSA controls (1.3±0.9% vs. 0.6±0.4% and 0.7±0.4%, respectively; P<0.05). Immunization of apoE-/- mice expressing GFP on the FoxP3 promoter showed no difference in Pent 5(+)CD8+FoxP3+ T cells. High fat diet feeding for 6 weeks did not affect Pent 5(+)CD8+ T cells compared to normal chow fed mice confirming specificity of the Pent 5(+)CD8+ T cell response to immunization. Pent 5 binding significantly reduced cytolytic activity of p210-immune CD8+ T cells compared to control (1.2±2.2% vs. 17.0±13.8%, respectively), indicating antigen-specific blocking. Pent 5(+)CD8+ T cells cultured for 21 days showed significantly higher cytolytic activity compared to Pent 5(-)CD8+ T cells (16.5±7.0% vs. 2.8±3.6%, respectively). Immunization with the p210 peptide reduced aortic atherosclerosis measured by en face oil red-o stain area compared to PBS and cBSA control groups (4.0±1.7% vs. 6.4±2.3% and 5.7±2.2%, respectively; P<0.01), confirming our previous report.

Conclusion:
The use of Pentamers provide clear evidence that p210 immunization results in CD8+ T cell activation with functions specific to the p210 antigen.
Abstract 351: MHC-II Tetramer-based Isolation of Atherosclerosis Autoantigen-specific T Cells
Authors: Gerhardt Teresa , Wolf Dennis , Kimura Takayuki , Jenkins Marc K , Dileepan Thamotharampillai , Ley Klaus
Published: June 2021

Background:
Immune cells, including T cells, figure prominently in atherosclerosis. T cells in the atherosclerotic plaque have been proposed to recognize self-antigen, such as peptides derived from Apolipoprotein B-100. However, antigen-specific T cells have not been identified in atherosclerosis. Here, we aimed to establish and optimize a novel MHC-II multimer based assay to detect and isolate T cells specifically recognizing the ApoB-100 amino residues 978-993. We have previously shown that vaccination with this peptide protects mice from atherosclerosis.

Materials and Methods:
We screened peptides from mouse ApoB-100 for high affinity binding to the mouse C57BL/6 MHC-II molecule I-Ab and designed a novel MHC-II-tetramer in order to detect CD4
+
T cells that specifically recognize one of these peptide epitopes (ApoB-100 978-993) by flow cytometry. This tetramer (P6:I-Ab) consists of four biotinylated MHC-II monomers that are linked by a streptavidin molecule and display the ApoB-100 978-993 peptide in the MHC-II binding grove. It was expressed in a drosophila expression system and subsequently labeled with PE or APC. T cells from mice immunized with the ApoB-100 978-993 peptide were isolated from spleen and lymph nodes, enriched for CD4
+
T cells by negative selection, labeled with a mixture of APC- and PE-labeled ApoB-100 978-993 tetramer and positively selected by magnetic beads to PE and APC. P6:I-Ab binding co-localized with CD4 in confocal microscopy. ApoB-100 978-993 P6:I-Ab
+
T cells showed higher expression of the activation marker CD44 than P6:I-Ab
-
T cells. When Nur77-GFP transgenic mice were immunized with ApoB-100 978-993, P6:I-Ab
+
CD4
+
T cells showed enhanced GFP signal by flow cytometry, indicating T Cell Receptor signaling. By optimizing these methods, we successfully increased the percentage and absolute number of P6:I-Ab
+
T cells by 50-100-fold compared to un-enriched samples. The negative control tetramer (2WIS) bound very few T cells.

Conclusion:
We have successfully established a method to isolate atherosclerosis autoantigen-specific T cells from mice immunized with ApoB-100 978-993.
Abstract 369: SMC-Specific Tgfbr1 Deficiency Inhibits Neointimal Hyperplasia In Injured Arteries
Authors: Liao Mingmei , Zhou Junmei , Yang Pu , Ali Yasmin , Wang Fen , Jiang Zhihua
Published: June 2021

Introduction:
Stented arteries and vein bypass grafts fail due to neointimal hyperplasia (NIH). Transforming growth factor (TGF)-β has been identified as a driving force for this pathology. Our previous studies suggest that TGFβ contributes to NIH via regulating cell kinetics at early stage and matrix metabolism at late stage. While this differential TGFβ effect may result from its signaling in different cell-groups, the dominant cellular effectors at each stage remain to be determined. In this study, we evaluated the effect of TGFβ signaling in smooth muscle cell (SMCs) on NIH in injured arteries.

Methods and results:
An inducible
Cre/loxP
system driven by a
Myh11
promoter was employed to delete SMC-specific
Tgfbr1
(

Tgfbr1
iko

). Male mice at 10 weeks of age were used in the study and those not carrying the
Cre
allele (

Tgfbr1
f/f

) served as controls. On the following day of the last tamoxifen injection (day 0, d0), animals received transmural mechanical injury to induce NIH in femoral arteries (FAs). By d28,

Tgfbr1
iko

attenuated NIH by 90% compared with

Tgfbr1
f/f

. However, the injured

Tgfbr1
iko

FAs contained fewer α-actin positive cells and displayed significantly higher rate of cell proliferation and apoptosis with less matrix deposition than

Tgfbr1
f/f

controls in neointima. Intense inflammatory infiltration was detected in adventitia of

Tgfbr1
iko

, but not

Tgfbr1
f/f

, FAs. To explore mechanisms underlying these cellular events, we examined the growth of SMCs explanted from

Tgfbr1
iko

and

Tgfbr1
f/f

aortas and assessed their response to TGFβ stimulation. As expected, TGFβ1 inhibited cell growth and induced expression of collagen and elastin in

Tgfbr1
f/f

SMCs. In contrast,

Tgfbr1
iko

SMCs were resistant to TGF-β1 stimulation, evidenced by insignificant change in levels of these variables. Intriguingly,

Tgfbr1
iko

SMCs grew significantly slower than

Tgfbr1
f/f

SMCs under standard culture condition. Daily refreshment of culture medium doubled the population-growth of

Tgfbr1
iko

, but not

Tgfbr1
f/f

, SMCs, indicating production of growth inhibitory factor(s) by

Tgfbr1
iko

SMCs.

Conclusion:
Blockade of SMC-specific TGFBR1 activity inhibits NIH in a short-term, but, the resultant phenotype may not favor long-term performance of the injured arteries.
Abstract 375: Calcification of Human Saphenous Vein Correlates With Endothelial Dysfunction: A Histopathophysiological and Morphological Study
Authors: Pedigo Sydney , Banathy Alex , Li Fan Dong , Cheung-Flynn Joyce , Brophy Colleen , Komalavilas Padmini
Published: June 2021

Background:
Endothelial dysfunction decreases nitric oxide, which leads to the development of intimal hyperplasia and, eventually, vein graft failure. Failed vein grafts have been found to have calcium deposits, enlarged intima, atherosclerotic plaques, and extracellular matrix deposition. This project examined the effect of calcification on endothelial function by measuring the endothelial relaxation and the expression of nitric oxide synthase (eNOS) in saphenous veins.

Methods:
Freshly collected HSV (58) samples from CABG patients were equilibrated in a muscle bath and phenylephrine-induced contraction and carbachol-induced relaxation (endothelium-dependent) were measured. Vein sections were stained using Verhoeff-Van Gieson (VVG), Movat, and Von Kossa stains. Endothelial coverage was determined by eNOS, and Lectin staining. Calcification, intimal and medial basal thickness, endothelial coverage, and extracellular matrix deposition data were quantified using light microscopy and digital image analysis.

Results:
Of the 58 veins analyzed, 17 displayed calcification. The average basal intimal thickness of the samples was 65.44 ± 7.99 μm. Presence of calcification in HSV correlated with higher intimal basal thickness (p=0.0092, n=46, panel A), lower endothelial relaxation (p=0.032, n=43, panel B), loss of eNOS expression (p=0.0397, n=29), loss of endothelial coverage (p=0.0178, n=29), and increased extracellular matrix deposition (p=0.0180, n=22, panel C).

Conclusions:
Calcified veins displayed lowered endothelial relaxation, higher intimal basal thickness, reduced endothelial coverage, reduced eNOS expression, and increased extracellular matrix deposition. Calcification was associated with factors that contribute to intimal hyperplasia formation and vein graft failure.
Abstract 391: Apolipoprotein E3, Apolipoprotein AI, and Apolipophorin III Chimeras Provide Insight Into the Domain Organization of Apolipoproteins
Authors: Horn James V , Lek Mark , Ibe Nnejiuwa , Ellena Rachel A , Beck Wendy H , Bielicki John K , Narayanaswami Vasanthy , Weers Paul M
Published: June 2021

Apolipoproteins (apo) A-I and E are exchangeable apolipoproteins that play a dominant role in regulating lipoprotein metabolism. They are composed of a series of amphipathic α-helices, which are organized into an N-terminal (NT) helix bundle domain and a smaller C-terminal (CT) domain. The objective of the current study is to understand the functional and structural role of the domains of these proteins by “domain swapping” using apoE3 and apoAI, both of which bear a 4-helix bundle in their NT domain, and apolipophorin III (apoLp-III), a monomeric one-domain model apolipoprotein. A series of chimeric apolipoproteins were generate: apoE3-NT/apoAI-CT, apoAI-NT/apoE-CT and apoLp-III/apoA-I-CT. The α-helical content of the chimeras was comparable to that of the parent proteins. Unfolding studies indicated that addition of CT-apoE3 to NT-apoAI conferred more stability to apoAI. While addition of CT-apoAI to NT-apoE3 had no significant effect on the stability of apoE3, addition of NT-apoAI to apoLp-III increased the stability of apoLp-III. Moreover, apoLp-III switched from a monomeric to an oligomeric protein upon addition of CT-apoAI, showing that CT-apoAI has a strong tendency to self-associate. Addition of CT-apoAI to NT-apoE or apoLp-III increased the lipid binding activity by ~ 10-fold, while addition of apoE-CT to apoA-I-NT had no measurable effect on the lipid binding activity. All three chimeras promote ABCA1-mediated cholesterol efflux from J774 macrophages, with the efflux capability being highest for apoAI-NT/apoE-CT. Whereas apoE3-NT/apoAI-CT elicits LDLr binding ability, apoAI-NT/apoE-CT did not. These results suggest that CT of apoAI is a strong promoter of lipid binding, while CT of apoE3 can improve cholesterol efflux ability of apoAI.
Abstract 397: Transgenic Expression of Cholesteryl Ester Transfer Protein in Female Mice Disrupts Liver and Plasma Triglyceride Metabolism With Estrogen Treatment in a Liver Network Involving Estrogen Receptor Alpha and Small Heterodimer Partner
Authors: Palmisano Brian T , Zhu Lin , Stafford John M
Published: June 2021

Background:
Triglycerides are an important risk factor for cardiovascular disease, especially in women. Hormone replacement therapy raises triglycerides (TG) in postmenopausal women, but mechanisms responsible remain poorly understood. Cholesteryl Ester Transfer Protein (CETP) is a serum protein that shuttles TG and cholesteryl ester between lipoproteins in humans and plays an important role in lipoprotein metabolism. The role of CETP in regulating whole body triglyceride homeostasis remains unclear. Mice (which naturally lack CETP) expressing a transgenic copy of CETP or their wild-type (WT) littermates were used to determine the role of CETP in regulating plasma and liver TG metabolism in response to estrogen.

Methods and Results:
Female CETP and WT mice were ovariectomized and given vehicle or estrogen treatment. Estrogen raised plasma TG in CETP females, but not in WT females. CETP expression impaired TG clearance independent of estrogen. Estrogen treatment raised very-low density lipoprotein (VLDL) TG production in CETP females, but not in WT females. Estrogen treatment augmented mRNA expression and protein activity of VLDL assembly and secretion targets (Apob, Mttp, PDI) in CETP females, but not in WT females. Additionally, CETP expression lowered liver TG content and increased mRNA expression of TG oxidation targets (Ppara, Acox1, Acadm, Cpt2). Deletion of liver estrogen receptor alpha prevented CETP-mediated decreases in liver TG content and increases in mRNA expression of TG oxidation targets. Liver-specific deletion of estrogen receptor alpha did not attenuate estrogen-mediated increases in plasma TG and VLDL-TG production in female mice expressing CETP. Deletion of liver small heterodimer partner attenuated CETP-mediated increases in plasma TG and VLDL-TG production with estrogen treatment. Deletion of small heterodimer partner also attenuated CETP-mediated increases in mRNA expression and protein activity of VLDL synthesis and assembly targets with estrogen treatment.

Conclusion:
These studies indicate several novel functions of CETP in regulating plasma and liver TG homeostasis. We also discovered that CETP expression in female mice also augments several aspects of estrogen action in liver that impact liver TG biology.
Abstract 4: Enhancing the Ability of Macrophages to Orchestrate Selective Autophagy and Lysosomal Biogenesis Protects Against Atherosclerosis
Authors: Sergin Ismail , Bhattacharya Somashubhra , Zhang Xiangyu , Evans Trent D , Dehestani Babak , Emanuel Roy , Razani Babak
Published: June 2021

The autophagy-lysosome system is a catabolic cellular mechanism that degrades dysfunctional proteins and organelles. The pro-atherogenic phenotype of mice with macrophage-specific autophagy deficiency (ATG5-/-) confirm the importance of this degradation system in the pathogenesis of atherosclerosis. The mechanistic basis appears to involve a two-step process in macrophages: the development of lysosomal dysfunction/membrane integrity by atherogenic lipids followed by an inability of lysosomes to handle and degrade cargo supplied by autophagy. A prominent sequelae of such blockage is the accumulation of cytoplasmic inclusions composed of polyubiquitinated protein aggregates and organelles which are normally targeted for selective autophagy by the protein chaperone p62. In order to stimulate the degradative capacity of macrophages, we developed mice with macrophage-specific overexpression of TFEB, a master transcriptional activator of both autophagy and lysosomal biogenesis. Macrophage TFEB ameliorated several deleterious effects of atherogenic lipids, namely the blunting of inflammasome activation, enhancing cholesterol efflux, accelerating the degradation of protein aggregates, and decreasing apoptosis. In vivo, macrophage TFEB overexpression reduced both plaque burden and plaque complexity in pro-atherogenic ApoE-/- mice fed a Western diet. Interestingly, TFEB’s atheroprotective effects were not only abrogated in the background of macrophage autophagy deficiency (ATG5-/-) but also in the background of p62-deficiency (p62-/-) suggesting the critical importance of selective autophagy and degradation of p62-enriched protein aggregates. Taken together, these data support the induction of a holistic pro-degradative response in macrophages (i.e. selective autophagy followed by lysosomal degradation) as a viable therapeutic strategy in atherosclerosis.
Abstract 402: miRNA Targeting of Oxysterol Binding Protein-like 6 (OSBPL6) Regulates Cholesterol Trafficking and Efflux
Authors: Ouimet Mireille , Hennessy Elizabeth , Koelwyn Graeme , van Solingen Coen , Hussein Maryem , Ramkhelawon Bhama , Rayner Katey , Garabedian Michael , Temel Ryan , Holdt Lesca , Tupser Daniel , Moore Kathryn
Published: June 2021

Cholesterol homeostasis is fundamental to human health, and is thus, tightly regulated. MicroRNAs exert potent effects on biological pathways, including cholesterol metabolism, by repressing genes with related functions. We reasoned that this mode of pathway regulation could be exploited to identify novel genes involved in cholesterol homeostasis. Here, we identify oxysterol binding protein-like 6 (
OSBPL6
) as a novel target of 2 miRNA hubs regulating cholesterol homeostasis: miR-33 and miR-27b. Characterization of
OSBPL6
revealed that it is transcriptionally regulated in macrophages and hepatocytes by LXR and in response to cholesterol loading, and in mice and non-human primates by western diet feeding.
OSBPL6
encodes the OSBPL-related protein 6 (ORP6), which contains a pleckstrin homology membrane-targeting domain and a FFAT domain predicted to interact with VAP proteins in the ER. Indeed, subcellular localization studies showed that ORP6 is associated with the plasma membrane, early endosomes, lysosomes and ER, suggesting a putative role for ORP6 in cholesterol trafficking between these compartments. Consistent with this, siRNA-mediated knockdown of
OSBPL6
results in aberrant clustering of endosomes and promotes the accumulation of free cholesterol in these structures. As a consequence, esterification of cholesterol at the ER is reduced, triggering activation of SREBP2. Conversely, ORP6 overexpression enhances cholesterol trafficking and efflux to apoA1 in macrophages and hepatocytes. Moreover, we show that hepatic expression of
OSBPL6
is positively correlated with plasma levels of HDL cholesterol in a cohort of 200 healthy individuals, whereas its expression is reduced in human atherosclerotic plaques. Collectively, these studies identify ORP6 as a novel regulator of cholesterol trafficking that is part of the miR-33 and miR-27b target gene networks that contribute to the maintenance of cholesterol homeostasis.
Abstract 407: Atherogenic Dyslipidemia in Patients with Diabetes Mellitus May Have Distinct High Density Lipoprotein Function
Authors: Song Wenliang , Du Jing , Qin Li , Zarich Stuart , Majumdar Sachin , Hwa John
Published: June 2021

There is growing evidence suggesting that high-density lipoprotein (HDL) function is more important than the HDL cholesterol (HDL-C) levels. HDL particles are highly heterogeneous and their function may also differ among different patient populations. There are two populations that are recognized tohave significant residual cardiovascular risk despite intensive statin therapy: atherogenic dyslipidaemia and diabetes mellitus. Atherogenic dyslipidemia and diabetes have significant homology with regards to lipid profiles. However, systemic epidemiological studies that describes the overlapping profiles between atherogenic dyslipidemia and diabetes is lacking. The function and types of HDL in these two populations needs to be carefully elucidated. HDL has many functions includingreverse cholesterol transport, anti-oxidant activity and anti-inflammatory activity. HDL may also exert “antiplatelet effects”but the mechanism is unclear.We assessed the prevalence of atherogenic dyslipidemia in diabetes mellitus using data from the National Health and Nutrition Examination Survery (NHANES) and data from Yale New Haven Health (YNHH). We found that atherogenic dyslipidemia and diabetes mellitushave overlapping lipid profiles but are clearly two distinct conditions. In our preliminary studies, we found that HDL inhibits platelet aggregation and activation. Very interestingly, we found HDL can induce mitochondria dependent platelet apoptosis. The antiplatelet effect of HDL, as observed by others in the past, may arise fromapoptotic platelets that do not aggregate, and thus the inhibition of platelet aggregation. Whether HDL subtypes from atherogenic dyslipidemia and diabetes patient have differential effects on platelet apoptosis are currently being investigated.
Abstract 421: N-acetylcysteine Attenuates Systemic Platelet Activation and Cerebral Vessel Thrombosis in Diabetes
Authors: Wang Bin , Aw Tak Y , Stokes Karen Y
Published: June 2021

Diabetes leads to higher risk for stroke and cerebral small vessel disease. Methylglyoxal (MG) is a metabolite of glucose that glycates proteins. We previously demonstrated that diabetes exacerbates stroke-induced brain injury, and that this correlates with brain methylglyoxal (MG)-to-glutathione (GSH). This was reversed by N-acetylcysteine. Here we tested if NAC could also protect against diabetes induced platelet activation and thrombosis. The streptozotocin (STZ)-induced mouse model of type 1 diabetes was used. Circulating blood platelet-leukocyte aggregates (PLAs) were analyzed by flow cytometry at 5 or 19 wks diabetes. Thrombus formation in venules and arterioles (pial circulation) was determined by intravital videomicroscopy one week later. Tail bleeding time was also measured at this time. NAC (2mM) was given in drinking water starting 3 wks before thrombosis assessment. As compared to Veh controls, # of PLAs were significantly increased by diabetes. This was primarily due to elevated lymphocyte-platelet aggregates at the early timepoint, whereas platelet-monocyte aggregates were significantly higher at the later timepoint. NAC decreased the formation of PLAs at both timepoints. At 6 wk diabetes, the onset of thrombus formation in cerebral venules and arterioles was accelerated, and time to complete occlusion of arterioles was shortened. Both the onset and cessation time in cerebral venules and arterioles were reduced at 20 wk diabetes compared with times in Veh mice. NAC significantly prolonged the thrombosis at both timepoints. Interestingly, the cessation times in arterioles and venules were shorter in the more advanced diabetic state than at 6 wks diabetes. The tail bleeding time was not changed at 6 wk, but significantly shortened at 20 wk. This was reversed by NAC treatment. Collectively, these results show that the diabetic blood and brain are more susceptible to platelet activation and thrombosis. Although the pro-thrombotic phenotype evolves with diabetes duration, NAC attenuate the thrombotic effects of diabetes. Future studies will determine if NAC is acting through its anti-oxidant capacity or by decreased dicarbonyl stress.
Abstract 422: Oxidation of Ldl by Iron at Lysosomal pH Leads to the Formation of Tryptophan Radicals Which Are Not Inhibited by Probucol
Authors: Ahmad Feroz , Leake David S
Published: June 2021

The LDL oxidation hypothesis proposes that cells oxidise LDL in arterial interstitial fluid and macrophages take it up rapidly, becoming foam cells. LDL oxidation is inhibited by interstitial fluid, however, and large clinical trials have shown no protection by antioxidants, including probucol. We therefore proposed that LDL might be nonoxidatively modified and aggregated by enzymes, such as sphingomyelinase, in interstitial fluid, rapidly phagocytosed by macrophages and oxidised by iron inside lysosomes, which have a pH of about 4.5 (Wen & Leake, 2007, Circ. Res.
100
, 1337). Tryptophan in apoB-100 might be involved in the initiation of LDL oxidation by copper (Giessauf
et al
., 1995, Biochim. Biphys. Acta
1256
, 221). We investigated the mechanisms of LDL oxidation by iron at lysosomal pH. LDL (50 μg LDL protein/ml) was oxidised by FeSO
4
or FeCl
3
(5 μM) at 37 °C in 150 mM NaCl/10 mM sodium acetate buffer, pH 4.5. Lipid oxidation was measured in terms of conjugated dienes at 234 nm and tryptophan oxidation by the loss of fluorescence (Ex/Em 282/331 nm).

Unexpectedly, both lipid peroxidation and loss of tryptophan fluorescence was faster with Fe
2+
than Fe
3+
. Interestingly, probucol did not inhibit lipid oxidation for about 100 min with Fe
2+
at pH 4.5 and did not prevent the loss of tryptophan fluorescence.In contrast, probucol inhibited entirely the initial oxidation of LDL by Cu
2+
at pH 7.4. Fe
2+
was completely consumed during 100 min of LDL oxidation at pH 4.5, as shown using bathophenanthroline and might generate superoxide radicals. Superoxide radicals would protonate at pH 4.5 to produce highly reactive and lipid-soluble hydroperoxyl radicals, which might abstract hydrogen atoms form lipids and tryptophan. As probucol would be expected to scavenge lipid radicals in the surface monolayer of LDL particles (Bard
et al
., 1994, J, Pharm. Pharmacol.
46
, 797), the initial oxidation of LDL at pH 4.5 would be expected to happen in the core of LDL where probucol has limited excess.

We propose the following mechanism for LDL oxidation by Fe
2+
at lysosomal pH.

Fe
2+
+ O
2
→ Fe
3+
+ O
2
•-

O
2
•-
+ H
+
↔ HO
2
•
(pK
a
4.8)

HO
2
•
+ TrpH → Trp
•
+ H
2
O
2

Trp
•
+ O
2
→ TrpOO
•

HO
2
•
+ LH → L
•
+ H
2
O
2

L
•
+ O
2
→ LOO
•

This might help to explain why probucol was not beneficial in the clinical trials.
Abstract 423: Potential Role of Natriuretic Peptides in Atherosclerosis and Inflammation
Authors: Aluganti Narasimhulu Chandrakala , Parthasarathy Sampath
Published: June 2021

Background:
Brain natriuretic peptide (BNP) is currently recognized as one of the important markers of Congestive heart failure (CHF). BNP levels are simple and objective measures of cardiac function in patients with ischemic injury. These measurements can be used to diagnose HF, including diastolic dysfunction and inflammation. Plasma levels of BNP and ANP are elevated in CHF, especially after myocardial infarction (MI). Furthermore, within the heart tissue, gene expression of BNP and ANP is reportedly up-regulated in animal models with MI and CHF, and in human heart disease. In this study, we tested the natriuretic peptides ability in reduction of peroxides as well as inflammation

Methods:
BNP and ANP were commercially synthesized and their ability to reduce lipid peroxides (LOOH) was measured using 13-hydroperoxyoctadecadienoic acid (13-HPODE). Low density lipoprotein (LDL) was isolated from human plasma and its oxidation was performed using copper in the presence or absence of the peptides. The ability of these peptides to neutralize charges of modified lipoproteins, as well as attenuate inflammation, were analyzed. Oxidized LDL (Ox-LDL)/Ac-LDL was pretreated with increasing concentrations of peptides to evaluate charge neutralizing properties of the peptides. RAW cells were incubated with LPS pretreated with peptides. RNA was isolated from treated cells and real time PCR was performed using mouse IL-1α and IL-6 primers.

Results:
Natriuretic peptides reduced 13-HPODE and inhibited the oxidation of LDL. Reduced IL-1α and IL-6 gene expression was observed in the presence of LPS.

Conclusion:
The results of our studies suggest that these peptides a) inhibit the oxidation of LDL and b) have an inhibitory effect on inflammatory cytokine/gene production in the presence of LPS. Based on these results, we predict that natriuretic peptides could have therapeutic potential in reducing CVD/atherosclerosis-associated inflammation.
Abstract 426: MicroRNA-30c Mimic Treatment Attenuates Hypercholesterolemia and Atherosclerosis in Mice
Authors: Irani Sara , Pan Xiaoyue , Peck Bailey , Iqbal Jahangir , Sethupathy Praveen , Hussain M.Mahmood
Published: June 2021

Background:
High plasma cholesterol levels are a major risk factor for atherosclerosis. Plasma cholesterol can be reduced by inhibiting lipoprotein production; however, this is associated with steatosis. Previously we showed that lentiviral mediated hepatic over-expression of microRNA-30c (miR-30c) reduced hyperlipidemia and atherosclerosis in mice without causing hepatosteatosis. Since viral therapy would be formidable, we tested the hypothesis that a miR-30c oligonucleotide mimic can mitigate hyperlipidemia and atherosclerosis without causing steatosis.

Methods and results:
Delivery of a miR-30c mimic to the liver diminished Western diet-induced hypercholesterolemia in C57BL/6J wild type mice. Reductions in plasma cholesterol levels were significantly correlated with increases in hepatic miR-30c levels. Long-term dose escalation studies showed that miR-30c mimic causes sustained reductions in plasma cholesterol without increasing hepatic lipids and plasma transaminases. Further, miR-30c mimic significantly reduced hypercholesterolemia and atherosclerosis in

Apoe
–/–

mice without causing steatosis or increasing plasma transaminases. Mechanistic studies indicated that miR-30c mimic reduced hepatic lipoprotein production, in part by reducing microsomal triglyceride transfer protein (MTP) activity. This is supported by the observation that miR-30c did not lower plasma cholesterol in liver-specific MTP deficient mice but did reduce hepatic lipid synthesis. In order to understand why there was no increase in hepatic lipids, we carried out whole transcriptome studies by RNA-sequencing. Bioinformatic analysis of the data revealed that genes down-regulated by miR-30c mimic are most significantly enriched for lipid synthesis pathways. Consistent with this finding, we showed that miR-30c mimic reduced fatty acid and glycerolipid synthesis in the liver but had no effect on hepatic β-oxidation.

Conclusions:
We conclude that miR-30c reduces plasma cholesterol by targeting MTP and prevents hepatosteatosis by repressing lipid synthesis programs in the liver. These findings suggest that increasing hepatic miR-30c levels may be a viable treatment modality for hypercholesterolemia and atherosclerosis.
Abstract 427: An Oral, Rising, Multiple-dose Tolerance, Pharmacokinetic, and Pharmacodynamic Study of Gemcabene in Healthy Volunteers
Authors: McShane Margaret , Radulovic Louis , Bisgaier Charles L
Published: June 2021

Background:
Gemcabene is a novel lipid-regulating compound being developed as an adjunct to diet and statin therapy for the treatment of dyslipidemia. Gemcabene was administered in a randomized, double-blind, rising, multiple-dose safety, tolerance, pharmacokinetic, and pharmacodynamic study in healthy subjects.

Methods:
Fifty healthy subjects (34 men and 16 women), with a mean age of 39.7 years and mean baseline LDL-C of 116.7 mg/dL were randomized (6:1) to either gemcabene 50, 150, 300, 450, 600/750, or 900 mg or placebo once daily for 29 days in six rising-dose cohorts.

Results:
Forty-seven subjects completed the study. Gemcabene significantly lowered total cholesterol by 18-21%, low-density lipoprotein-cholesterol (LDL-C) by 26-32%, and apolipoprotein B (apoB) by 8-21% from both baseline and placebo at the 450, 750/600, and 900 mg doses (
Table below
). Gemcabene was rapidly absorbed with dose proportional increases in maximum plasma concentration (Cmax) and area under plasma concentration time curve from 0 to 24 hours (AUC
(0-24)
) values. Elimination half-life values were independent of dose and averaged 32-41 hours. Multiple doses of gemcabene up to 900 mg for 29 days were generally well-tolerated. There were no treatment-related serious adverse events. Adverse events reported were generally mild to moderate in intensity.

Conclusion:
Gemcabene exposure increased proportional with dose, was well tolerated, and was observed to significantly lower total cholesterol, LDL-C, and apo B at once daily doses of 450 to 900 mg. Gemcabene is being developed as an oral, lipid-altering agent to be used as an adjunctive therapy for the treatment of dyslipidemia, including the rare indication of HoFH.

Clinical Implications:
Gemcabene is being developed as a potential treatment for dyslipidemia.
Abstract 43: Prothrombinase Assembled with Factor V Leiden is Resistant to Inhibition by Tissue Factor Pathway Inhibitor α Lowering the Procoagulant Threshold for Initiation of Coagulation
Authors: Wood Jeremy P , Baumann Kreuziger Lisa M , Maroney Susan A , Camire Rodney M , Mast Alan E
Published: June 2021

Factor V (FV) assembles with factor Xa (FXa) into prothrombinase, the enzymatic complex that converts prothrombin to thrombin. Tissue factor pathway inhibitor α (TFPIα) inhibits prothrombinase by high affinity interactions with FXa-activated FV and the FXa active site, thereby blocking the initiation of coagulation. FV Leiden (FVL) is strongly linked to venous thrombosis through its resistance to degradation by activated protein C (aPC), which enhances the propagation of coagulation. FVL combined with a 50% reduction in TFPI causes severe thrombosis and perinatal lethality in mice, suggesting that FVL also promotes the initiation of coagulation. To examine this possibility, thrombin generation assays initiated with limiting FXa were performed with control or FVL plasma and platelet-rich plasma (PRP). The activation threshold for thrombin generation was 10 to 20 pM FXa in 10 control plasmas, but was 5 pM in 4 of 10 homozygous FVL plasmas. FVL PRP had a similar decrease in the activation threshold. The differences in activation threshold were totally normalized by an anti-TFPI antibody, while exogenous TFPIα and a FV-binding peptide that mimics TFPIα had reduced anticoagulant activity in FVL plasma, revealing that the procoagulant effects of FVL in these assays rely on TFPIα. Next, FVL plasmas were studied in fibrin clot formation assays, as they are sensitive to small amounts of thrombin. In reactions activated with 0.5 pM FXa, 1 of 8 control plasmas, compared to 7 of 8 homozygous FVL plasmas, clotted within 60 minutes, with differences again normalized by the anti-TFPI antibody. In prothrombinase activity assays using purified proteins, TFPIα was a 1.7-fold weaker inhibitor of prothrombinase assembled with FVL compared to FV. Thus, in addition to its aPC-mediated effect on the propagation of coagulation, FVL is resistant to TFPIα inhibition, exerting a procoagulant effect on coagulation initiation. This is evident in responses to small stimuli, where TFPIα blocks clotting in plasmas with FV but not FVL. The TFPIα-mediated modulation of the procoagulant threshold may explain the severe perinatal thrombosis in FVL mice with decreased TFPI and be clinically relevant in the clotting associated with oral contraceptives, which cause acquired TFPI deficiency.
Abstract 432: Endothelial Cells Express miR-33a-5p and Release Extracellular Vesicles Containing miR-33a-5p
Authors: Stamatikos Alexis , Vojtech Lucia , Dronadula Nagadhara , Dichek David
Published: June 2021

Background:
The microRNA miR-33a-5p increases cellular cholesterol by posttranscriptional silencing of genes involved in cholesterol efflux. Previous studies showed that miR-33a-5p inhibition protects against atherosclerosis. Atheroprotective effects of miR-33a-5p inhibition are attributed to actions in hepatocytes that raise plasma HDL and actions in macrophages that enhance cholesterol efflux. We hypothesized that miR-33a-5p is also expressed in primary arterial endothelial cells (EC) and that miR-33a-5p might contribute to atherosclerosis by promoting cholesterol accumulation in EC. We also hypothesized that EC could release miR-33a-5p in extracellular vesicles (EV; including exosomes and microvesicles) that transferred miR-33a-5p to neighboring vascular cells such as SMC and macrophages, inhibiting cholesterol efflux, promoting lipid accumulation in these cells, and accelerating atherosclerosis.

Methods:
We cultured bovine aortic endothelial cells (BAEC) in serum-free medium, collected the medium, and isolated EV by ultracentrifugation. To test whether we had isolated EV, we compared EV and BAEC lysates by SDS-PAGE, Coomassie blue staining, and immunoblotting. We measured EV size with nanoparticle tracking analysis and imaged the particles with transmission electron microscopy. We extracted total RNA from BAEC and EV, measured RNA size using BioAnalyzer, and detected miR-33a-5p expression using RT-PCR and restriction digestion as well as qRT-PCR.

Results:
Coomassie blue staining revealed large differences between BAEC and EV lysates. The exosome marker CD9 was present at higher levels in EV lysate compared to BAEC lysate. Most of the EV were in the size range of exosomes, and appeared as typical lipid membrane-bound spheres by electron microscopy. EV contained primarily small RNA (~25-200 nucleotides). miR-33a-5p was detected both in BAEC lysates and in detergent-soluble EV.

Conclusions:
Primary arterial EC express miR-33a-5p and release EV rich in small RNA, including miR-33a-5p. Manipulating miR-33a-5p expression in EC, for example with a miR-33a-5p antagomiR, may be an effective therapeutic approach for increasing cholesterol efflux from multiple vessel wall cell types and preventing/reversing atherosclerosis.
Abstract 434: LDL-like Particles in HDL Density Have Small Diameters and Stimulate Macrophages Similar to LDL
Authors: Maekawa Ysutaka , Takase Hiroshi , Ito Yasuki , Lund-Katz Sissel , Tsujita Maki
Published: June 2021

We have been detected small portion of LDL-like lipoprotein in HDL density (1.063 <d< 1.21 g/mL) from human plasma. This β-migrating lipoprotein has affinity to Dextrin Sulfate (DS) column of which use for LDL apheresis (LA-15, Kanenka Medix Corp, Japan). Here, we isolated and examined character of this human dense LDL (d-LDL) isolated by ultracentrifugation and DS column then evaluated its potential on mouse macrophages foam cell development. Protein composition of d-LDL fraction detected by SDS-PAGE was similar to LDL fraction rather than HDL
DS
(remaining after DS column). This d-LDL particle size was measured by Transmission Electron Microscopic-negative staining method (JEM-1011J, x200,000) and this digital image data were further analyzed by Developer Toolbox
TM
(GE Healthcare) for particle area and diameter. The calibration speed of average 420.4 lipoprotein particles per 2440 x 3571 pixels image was less than 10 sec by this program. The d-LDL contained smaller particles compared to the LDL fraction, significantly. Small dense LDL (sdLDL) detected by DENKA assay system revealed increased sdLDL/LDL ratio in HDL
DS
fraction than d-LDL or LDL fractions. Perhaps, the sdLDL has low affinity to the DS column. In the other hand, Lp(a) was highly bound to DS column. The acetylated LDLs (ac-LDL, ac-d-LDL) were load on mouse peritoneal resident macrophage for 48 hrs to produce the foam cells. Both acetylated LDLs successfully induced foam cells while cellular choline labeled phosphatidylcholine was twice as high as in ac-d-LDL treated foam cells. The cholesterol content in the macrophage was also increased by ac-d-LDL. d-LDL fraction was re-isolated by “masked” DS column from non-specifically bound plasma proteins (d-LDL
ri
). Difference of phospholipid synthesis during macrophages foam cell development was abolished between by ac-LDL and by ac-d-LDL
ri
. Thus, original DS-Cellulose column bounds LDL but also inducer/inducers of macrophage lipid synthesis from human plasma HDL fraction. These results indicate LDL-like lipoprotein in HDL fraction, d-LDL, has smaller diameter than LDL. The acetylated d-LDL
ri
activated phospholipid synthesis in mouse peritoneal macrophage as the same extent to Ac-LDL.
Abstract 445: Endothelial Inflammation and Loss of Bone Morphogenetic Protein Receptor 2 in Oscillatory Shear Stress Model
Authors: Ezeuka Collins
Published: June 2021

Background:
Bone Morphogenetic Protein Receptor II (BMPR2) plays an unexpected role as a critical anti-inflammatory and anti-atherogenic protein in endothelial cells (ECs) via a reactive oxygen species (ROS) and NFκB-dependent mechanism. Pro-atherogenic stimuli such as disturbed laminar flow, angiotensin II, hypercholesterolemia and the pro-inflammatory cytokine TNFα, significantly downregulate BMPR2 expression in endothelium, while anti-atherogenic stimuli such as laminar flow and statins upregulate BMPR2’s expression in vivo and in vitro. These findings suggest that there may be a common mechanism by which pro-atherogenic factors downregulate BMPR2 expression and that protecting or restoring its expression could be a novel therapeutic approach for prevention and treatment of atherosclerosis. Our preliminary studies have identified microRNAs that possibly play a causative role in the loss of BMPR2, by binding to its 3’-UTR, leading to degradation of BMPR2, endothelial dysfunction, inflammation, and subsequent atherosclerosis.

Hypothesis:
Rescuing loss of BMPR2 will decrease endothelial inflammation and atherosclerosis

Methods:
Our in vitro model of disturbed blood flow is characterized by a cone and plate system, wherein mouse aortic endothelial cells are subjected to unidirectional laminar shear (LS 15 dyn/cm2) or oscillatory shear (OS, +/1 5 dyn/cm2 at 1 Hz frequency) for 24 hours. Endothelial cell inflammatory markers, BMPR2, and specific microRNA mRNA transcript fold changes, were then assessed via qPCR.

Results:
Under oscillatory flow conditions, in our in vitro shear stress system, BMPR2 is lost and mouse aortic endothelial cells acquire an inflammation phenotype, with a corresponding increase in the fold change of mRNA for microRNAs-17, 21, 25, and 181.

Conclusion:
We have identified microRNAs that may target BMPR2, leading to its degradation, and subsequent onset of endothelial inflammation. Blocking the aforementioned microRNAs may represent a novel therapy in the treatment of endothelial inflammation and subsequent atherosclerosis.
Abstract 450: Modification of HDL by Reactive Aldehydes Impairs HDL's Athero-protective Functions
Authors: Holme Rebecca L , Chadwick Alexandra C , Proudfoot Sarah C , Chen Yiliang , Ramakrishnan Devi Prasadh , Silverstein Roy L , Sahoo Daisy
Published: June 2021

High density lipoproteins (HDL) are athero-protective particles that promote the removal of excess cholesterol from lipid-loaded macrophages and stimulate their migration in order to protect against foam cell formation, a precursor to atherosclerotic plaque build-up. Recently, studies have shown that oxidative modification of HDL prevents HDL from protecting against atherosclerosis; however, the exact mechanisms by which this occurs are not well defined. We hypothesize that oxidative modification of HDL by reactive aldehydes such as acrolein (a major component of cigarette smoke) and 4-hydroxynonenal (HNE; a product of lipid peroxidation) impairs HDL’s athero-protective effects in macrophages. We tested our hypothesis using three different assays. First, we determined that modified forms of HDL upregulate mRNA levels of pro-atherogenic scavenger receptors such as cluster of differentiation 36 (CD36), a known oxidized LDL receptor. Incubation of macrophages with native HDL did not exert similar effects. Second, we tested the ability of oxidized HDL to prevent foam cell formation. Peritoneal macrophages isolated from WT C57Bl/J mice were cholesterol-loaded and incubated with native HDL, acrolein-modified HDL (acro-HDL), or HNE-modified HDL (HNE-HDL). Oil Red-O staining demonstrated that 24% of macrophages had foam cell formation upon incubation with native HDL, whereas 61% and 49% foam cell formation was observed for acro- and HNE-HDL, respectively. Preliminary data suggests this may be CD36-dependent. Finally, using a Boyden chamber assay, we demonstrated that both acro- and HNE-HDL, but not native HDL, had an impaired ability to promote macrophage migration (43% and 72% of HDL cell migration levels, respectively). We determined that the inability of acro- and/or HNE-HDL to stimulate macrophage migration may be due to an impaired ability of these modified lipoproteins to activate the PI3K pathway, as shown by decreased levels of phosphorylated protein kinase B (Akt). In conclusion, we have identified three independent mechanisms by which modification of HDL with acrolein or HNE impairs HDL’s cardio-protective effects and, instead, generates a particle that promotes pathways that lead to atherosclerosis.