bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 222: Dkk1 and Msx2-Wnt7b Signaling Reciprocally Regulate the Endothelial-Mesenchymal Transition in Aortic Endothelial Cells
Authors: Cheng Su-Li , Shao Jian-su , Behrmann Abraham , Krchma Karen , Towler Dwight A
Published: July 2021

Objective

Endothelial cells (ECs) can undergo an endothelial-mesenchymal transition (EndMT) during tissue fibrosis. Wnt- and Msx2-regulated signals participate in arteriosclerotic calcification and fibrosis. We studied the impact of Wnt7, Msx2, and Dkk1 (Wnt7 antagonist) on EndMT in primary aortic endothelial cells (AoECs).

Methods and Results

Transduction of AoECs with vectors expressing Dkk1 suppressed EC differentiation and induced a mineralizing myofibroblast phenotype. Dkk1 suppressed claudin 5, PECAM, cadherin 5 (Cdh5), Tie1 and Tie2. Dkk1 converted the cuboidal cell monolayer into a spindle-shaped multilayer and inhibited EC cord formation. Myofibrogenic and osteogenic markers - e.g., SM22, type I collagen, Osx, Runx2, alkaline phosphatase – were upregulated by Dkk1 via activin-like kinase / Smad pathways. Dkk1 increased fibrosis and mineralization of AoECs cultured under osteogenic conditions - the opposite of mesenchymal cell responses. Msx2 and Wnt7b maintained the “cobblestone” morphology of differentiated ECs and promoted EC marker expression. Deleting EC Wnt7b with the Cdh5-Cre transgene in Wnt7b(fl/fl);LDLR-/- mice upregulated aortic osteogenic genes (Osx, Sox9, Runx2, Msx2) and nuclear pSmad1/5, and increased collagen accumulation.

Conclusions

Dkk1 enhances EndMT in AoECs, while Msx2-Wnt7 signals stabilize EC phenotype. EC responses to Dkk1, Wnt7b, and Msx2 are the opposite of mesenchymal cell responses, coupling EC phenotypic stability with osteofibrogenic predilection during arteriosclerosis.
Abstract 247: Association of Soluble RAGE Levels with Carotid Atherosclerosis: The Northern Manhattan Study (NOMAS)
Authors: Hudson Barry I , Gardener Hannah , Liu-Mares Wen , Dong Chuanhui , Cespedes Sandino , Elkind Mitchell S , Wright Clinton B , Sacco Ralph L , Rundek Tatjana
Published: July 2021

Introduction

The Receptor for Advanced Glycation End-products (RAGE) is a multi-ligand receptor that propagates vascular cell dysfunction leading to proinflammatory disease states. RAGE is produced as a membrane bound and soluble isoform (soluble RAGE (sRAGE)), with the soluble isoform demonstrated to act as a RAGE decoy preventing cellular activation and atherosclerosis. Recent cohort studies have suggested that serum levels of sRAGE are associated with the risk of CVD and therefore may be a novel biomarker for cardiovascular disease states.

Hypothesis

We hypothesized that sRAGE levels are associated with subclinical atherosclerosis in an ethnically diverse population.

Methods

We included 1,102 stroke-free participants from the multi-ethnic Northern Manhattan Study (NOMAS) who underwent high-resolution carotid B-mode ultrasound to measure carotid plaque phenotypes (density, thickness, and area) and carotid intima-media thickness (IMT). Plaque density was characterized by Gray Scale Median (GSM). Serum sRAGE was measured by ELISA and log-transformed to stabilize variance. Multiple linear and logistic regressions were employed to estimate sRAGE associations with IMT and plaque measures.

Results

The mean age at time of ultrasound was 70.7±8.6yrs; 65% were Hispanic, 19% black, and 16% white. The majority of subjects had carotid plaque present (54%) with the median GSM 38(0-190). Mean plaque thickness (IQR) was 1.30(0-3.99)mm and mean area (IQR) 2.43(0-96.75)mm2. Mean IMT was 0.93±0.09mm. High sRAGE levels were associated with more echolucent plaques (OR 1.2, 95% CI 1.03-1.42), especially among Hispanics (OR 1.26, 95% CI 1.04-1.54). These relationships remained after adjusting for sociodemographic and vascular risk factors. No association was seen between sRAGE levels and carotid IMT, plaque thickness or area.

Conclusion

In the present study, higher sRAGE levels were associated with echolucent (lower density) plaque, especially among Hispanic subjects. Our data suggest sRAGE levels may be associated with atherosclerotic plaque morphology and its vulnerability, especially among minority groups. These findings further support RAGE as a novel target for anti-atherosclerosis interventions.
Abstract 268: Revascularization with Open Bypass vs. Angioplasty and Stenting of the Lower extremity Trial (ROBUST)
Authors: Malas Mahmoud B , Qazi Umair , Black James , Reifsnyder Thomas , Freischlag Julie , Perler Bruce
Published: July 2021

Background

Peripheral Arterial Occlusive Disease (PAD) is the most common vascular condition affecting 3-10% of the general population, and as high as 15-20% of those over 70 years of age. PAD is also a major marker for cardiovascular disease and stroke.

The Transatlantic Inter-Society Consensus (TASC II) classified superficial femoral artery lesions into 4 categories based on length and extent of disease. For TASC II A (short) lesions angioplasty with stenting (PTA/S) has shown to be effective. For TASC II D (long occlusion) open bypass (OB) is superior to PTA/S in patency and long term outcome. However for TASC II B and C (moderate) lesions there are conflicting reports on the best treatment modality. ROBUST is the first randomized clinical trial comparing OB and PTA/S in TASC II B and C lesions.

Primary objective

Patency (<50% restenosis on duplex), re-intervention rate.

Secondary objectives

Quality of life improvement, morbidity and mortality rate.

Methods:/Study Population

Patients with intermittent claudication who fail medical management or patients with critical limb ischemia are eligible. Patients are maximized medically. Patients with TASC II B or C lesions are prospectively randomized into either OB or PTA/S group; patients with TASC A and D lesion are non-randomized receiving PTA/Stent or Bypass respectively. Patients are followed at 1, 6 and 12 month and yearly up to five years with physical exam, ABI, duplex and QOL questionnaire.

Study Status

Study approved by Hopkins IRB. The study is actively enrolling participants, and making steady progress. Last DSMB Approval on 10/31/12.

ClinicalTrials.gov
Identifier

NCT01602159

Results

Total Consented = 29, Total Enrolled = 22, Screen fail = 7, Withdrawn = 0, Death = 1, TASC A = 4, TASC B = 7, TASC C = 1, TASC D = 10, Total randomized = 8

Patency

One patient with TASC A (non-randomized PTA/S group) and one TASC B (randomized to OB) had restenosis. Both restenosis were treated successfully with angioplasty.

Mortality

No intra operative death reported. One unrelated death at 6-month (TASC B lesion randomized to PTA/S)

Discussion/Significance of Impact

Providing level-1 evidence, ROBUST will establish treatment guidelines in such lesions and eliminate un-necessary procedures and reduce health care costs
Abstract 28: Carboxyalkyl-pyrrole Phosphatidylethanolamine Adducts Are Present In Vivo And Induce Platelet Activation Via Toll-like Receptor
Authors: Biswas Sudipta , Panigrahi Soumya , Zimman Alejandro , Podrez Eugene
Published: July 2021

A prothrombotic state and increased platelet reactivity are common in pathophysiological conditions associated with oxidative stress. Lipid peroxidation, a major consequence of oxidative stress generates highly reactive products capable of modifying autologous proteins as well as lipids. Hydroxy-ω-oxoalkenoic acids and their carboxyalkylpyrrole (CAP) protein adducts are recently described products of lipid peroxidation with strong biological activity mediated by Toll like receptors (TLR). Phosphatidylethanolamine (PE) is the second most abundant phospholipid in the living organisms. While recent studies suggest that PE is a major target for covalent modification by reactive products of lipid peroxidation, the presence of such products in vivo, their biological activities and receptors involved are not established. We now report that CAP-PE adducts are present in vivo in circulation and are significantly elevated in plasma of hyperlipidemic apoE-/- mice. In vitro experiments demonstrated that CAP-PE adducts induce platelet integrin αIIbβ3 activation, P-selectin expression and promote platelet aggregation. Multiple complimentary approaches demonstrated that platelet activation by CAP-PE is mediated by TLR2 and TLR1. Furthermore, direct interaction of CAP-PE and TLR2 was demonstrated. CAP-PE induced assembly of TLR2/TLR1 receptor complex in platelets leading to downstream signaling via MyD88/TIRAP-dependent pathway. CAPs-PE induced signaling included phosphorylation and activation of IRAK4 and subsequent activation of TRAF6, Src family kinase, Syk and PLCγ2. Thus, our study identified carboxyalkylpyrrole adducts of phosphatidylethanolamine as novel end products accumulating in circulation in hyperlipidemia that can induce platelet activation via innate immunity signaling pathway.
Abstract 290: Microrna-199a and Glut1 are Differentially Expressed in Primary Human Arterial and Venous Endothelium and are Deregulated in Culture
Authors: Richardson Matthew R , Lai Xianyin , Yoder Mervin C
Published: July 2021

Introduction

Studies over the last decade have established that the identity of arterial and venous endothelial cells (ECs) is governed by both genetic and environmental factors. Although the ephrin/Eph system is known to be a key determinant of that identity, it is unlikely that system is solely responsible for all the differences between arterial and venous ECs. Furthermore, microRNAs are becoming increasingly recognized for their important role in regulating gene expression and cell fate. We investigate here novel potential markers and regulators of arterial EC identity and the effects of environmental cues on these molecules.

Methods

ECs were freed from the basement membrane of human umbilical arteries and veins (HUAECs, HUVECs) (n=6) by enzymatic digestion and purified by flow cytometry (CD31+, CD45-) prior to lysis and quantitative PCR or analysis using HPLC-MS/MS. Protein quantification was performed using a proprietary software package (IdentiQuantXL
TM
). HUVECs and HUAECs were harvested directly from umbilical cords and placed directly into collagen coated 6-well plates and cultured in standard conditions in EGM2.

Results

We found that microRNA-199a levels were 23 fold higher in HUVECs than HUAECs (p=0.0001). One of microRNA-199a’s theoretical downstream targets is GLUT1, a facilitative glucose transporter thought to be responsible for basal glucose uptake. GLUT1 mRNA and protein levels were significantly higher (5.5 and 4.3 fold, respectively) in HUAEC than HUVECs (p=0.00002). Using tissue immunohistochemistry (IHC) of we confirmed that GLUT1 expression is restricted to arterial ECs in human umbilical cord and adult peripheral artery and vein sections. Finally, GLUT1 mRNA levels are 2 fold higher in HUAECs than HUVECs in culture at P2 and levels decrease until P5 when they become equal indicating that culture has a homogenizing effect on endothelial heterogeneity.

Conclusions

MicroRNA-199a levels are higher in HUVECs than HUAECs and GLUT1, its downstream target, is higher in HUAECs both at the mRNA and protein level. Protein expression is restricted to arteries in both the new born umbilical cord and adult peripheral vessels. Importantly, expression in both cell types is downregulated and equilibrated by cell culture.
Abstract 297: Cyclic GMP Reduces Vascular Smooth Muscle Migration through Inhibition of TGF-ß1/Smad Signaling
Authors: Vuncannon Jackson R , Stone Joshua D , Martin Danielle N , Joshi Chintamani N , Adderley Shaquria P , Tulis David A
Published: July 2021

Abnormal vascular smooth muscle (VSM) growth remains an elemental foundation of vasculoproliferative disorders including atherosclerosis and restenosis. Many second messenger, cytokine, and growth factor signals mediate control of VSM growth, and among these is transforming growth factor (TGF)-β1, a pluripotent cytokine with wide-ranging yet often opposite effects in VSM. Cyclic nucleotide signaling also exerts powerful growth control of VSM, and our previous work has helped establish a biological link between cyclic GMP and TGF-β1 in injured carotid arteries. The current study characterized the influence of cyclic GMP on TGF-β1 and its receptor-activated Smad3 in rat primary VSM cells. The heme-dependent soluble guanylate cyclase (sGC) stimulator BAY 41-2272 (BAY41) significantly increased cyclic GMP and site-specific phosphorylation of vasodilator-activated serum phosphoprotein (VASP) in manner indicative of active protein kinase G (PKG) and PKA signaling. Recombinant TGF-β1 (10 ng/ml) significantly stimulated phospho-Smad3 (Ser
423/425
) and decreased inhibitory Smad7 in VSM cell homogenates, and using flow cytometry significantly increased cells in G
2
/M and expression of cyclins D and E and Cdk2 and Cdk4 while decreasing expression of inhibitory p21 and p27 after 24 hours compared to vehicle controls. TGF-β1 also significantly increased cell numbers compared to controls after 48 hours, thus confirming growth promoting capacities of TGF-β1 in VSM. In cell lysates double-sandwich ELISA revealed that BAY41 significantly reduces total and active TGF-β1, and Western analyses showed it significantly decreases total and phospho-Smad3
Ser423/425
expression and reduces MMP-2 and MMP-9 expression and activity (via column zymography) in both cell lysates and conditioned media after 1 and 48 hours. BAY41 also significantly reduced serum- and PDGF-stimulated cell migration between 6 and 18 hours using an in vitro scrape injury and a transwell assay. In comparison, inclusive effects of BAY41 were replicated by its prototype YC-1 and by the heme-independent sGC activator BAY 60-2770. These data clearly support growth protective capacities of cGMP in VSM and propose it operates through attenuation of TGF-β1/Smad3 signaling.
Abstract 363: Proatherogenic and Proinflammatory Effects of Angiotensin II are Mediated by Activation of HIF-CD36 Pathway in Macrophages
Authors: Ren Fu , Chadipiralla Kiranmai , Raij Leopoldo , Zhou Ming-sheng
Published: July 2021

Recent studies have demonstrated that the renin angiotensin system (RAS) has profound effects on atherosclerosis, but the mechanisms underlying remain unclear. Macrophages play a central role in atherogenesis through scavenger receptor-mediated lipid accumulation, production of inflammatory cytokines and transformation of macrophages into foam cells. Macrophages express all components of RAS. Here we investigated the effects of Ang II on macrophage activation and foam cell formation, and underlying the mechanisms involved. In human THP1 macrophages, Ang II (100 nM) in time-dependent manner increased the mRNA expression of proinflammatory cytokines MCP1, TNFa, IL1b, IL6, CXCL9 and PAI1, Ang II also upregulated mRNA expression of CD36, TLR4 and hypoxia induced factor (HIF)1a. Knockdown of HIF1a by SiRNA prevented Ang II upregulation of CD36 and TLR4 expression, and significantly reduced the Ang II-induced proinflammatory cytokine expression. Next, we investigated the effects of Ang II on oxLDL uptake and foam cell formation, Ang II increased oxLDL uptake, which was prevented by either siRNA HIF1a or siRNA CD36. The incubation with oxLDL (50 ug/ml) for 72 hours result in lipid deposition in macrophage or foam cell formation, preincubation with Ang II further increased oxLDL-induced lipid deposition and foam cell formation, which was also prevented by either siRNA HIF1a or siRNA CD36. Infusion of Ang II into Apo E-/- mice significantly increased systolic blood pressure, the peripheral blood CD11b+/LyC6Hi inflammatory monocyte population and mRNA expression of IL1b and TNF1a in the peritoneal macrophages, Ang II also significantly increased atherosclerotic lesion formation. Hydrolazine normalized SBP but did not significantly reduce CD11b+/LyC6hi inflammatory monocyte population and atherosclerotic lesion formation in the apo E-/- mice treated with Ang II. The present study demonstrates, for the first time, that Ang II activates HIFa-CD36 pathway in human macrophage, which potentiate atherogenic lipid-induced macrophage activation and foam cell formation. Our novel findings may elucidate new mechanisms by which Ang II promotes atherogenesis through mediating macrophage activation and lipid metabolism.
Abstract 368: Family History of Premature Cardiac Events and Sub Clinical Atherosclerosis in Children and Young Adults: A Systematic Review
Authors: Sinha Pragya , Rana Jamal S , Oni Ebenezer T , Aneni Ehimen C , Blumenthal Roger S , Agatston Arthur A , Blaha Michael J , Blankstein Ron , Santos Raul , Nasir Khurram
Published: July 2021

BACKGROUND

The association between a positive family history (PFH) of premature cardiovascular disease (PCVD) and atherosclerosis has been explored in numerous studies. In adults, various studies have confirmed a significant positive correlation between a PFH and PCVD. Scant literature however, focuses on young individuals. Nevertheless, it is important to understand the impact that a PFH has in young people because the foundations of atherosclerosis and adverse cardiac behaviors develop in youth. In this paper, we aimed to systematically review the evidence linking a PFH of PCVD to indirect markers of subclinical atherosclerosis.

METHODS

The search was conducted on Medline, Web of Science and Embase. ‘Family history’, ‘children/young adults’ and ‘subclinical atherosclerosis’ were the three main concepts used. Increase in mean carotid IMT (cIMT), endothelial dysfunction and vascular inflammation were used as indirect measures of subclinical atherosclerosis.

RESULTS

1191 articles were identified in the initial search. 24 papers with 5400 participants were included in the final review. There were five cohort studies and nineteen case control studies from twelve countries. Mean cIMT was found to be significantly increased in those with a PFH by eleven of the fourteen papers reviewed. Endothelial dysfunction, measured by flow mediated dilatation (FMD), was found to be significantly increased in five of the seven included studies. The evidence on vascular inflammation was somewhat inconsistent with only ten of the nineteen studies demonstrating significance. The results tend to suggest that an elevated mean cIMT, as well as a greater degree of endothelial dysfunction are seen in children and young adults with a PFH of PCVD. Moreover, these differences exist in asymptomatic children as young as 8-9 years (4 studies) in the absence of any other cardiac risk factor.

DISCUSSION

Individuals with a PFH of PCVD have evidence of subclinical atherosclerosis in their youth demonstrating an accelerated tendency to acquire cardiovascular disease. Some of this risk may be attributable to behavioral risk clustering in families. However, a significant proportion of this elevated risk is related solely to a positive family history and needs attention.
Abstract 373: The Association of 9p21.3 Locus with Coronary Atherosclerosis: A Systematic Review and Meta-Analysis
Authors: Munir Muhammad S , Wang Zhen , Steffen Mark W , Erwin Patricia J , Murad Mohammad H , Kullo Iftikhar J
Published: July 2021

Background

The association between the 9p21-3 locus and coronary heart disease (CHD) is well known. We performed a systematic review of the literature to assess whether this locus was associated with severity of coronary atherosclerosis.

Data Sources

Multiple relevant electronic databases were searched through September 2012 for English language studies with comparative design.

Data extraction

We tested the association of the 9p21-3 locus: 1) measures of severity of coronary atherosclerosis [number of diseased vessels, Gensini Score, Duke CHD Prognostic Index (Dc.PI)]. 2) angiographic outcomes [change in minimum lumen diameter ([[Unable to Display Character: ∆]]MLD) and number of new lesions at follow-up] and 3) key clinical outcomes (all-cause mortality, recurrent myocardial infarction and need for coronary revascularization). Genotypes were classified either as homozygous low risk (Lo) or homozygous high risk (Hi).

Data Synthesis

Relative risks (RR) were pooled using the random effects model. Seventeen distinct data sets that included 15,826 participants were analyzed. Ten studies (n=9807) reported number of diseased vessels at baseline. Hi genotype was associated with a 33.7% increased risk of triple vessel disease (95% CI 1.08-1.65; p=0.01). The Hi genotype was associated 0.27 higher baseline Gensini Score (95% CI 0.04-0.48; p=0.01) in 3787 individuals. The locus was not associated with Dc.PI [RR=0.21 (95% CI -0.14-0.55; p=0.25)]. The high risk genotype did not predict [[Unable to Display Character: ∆]]MLD or number of new lesions at follow-up. There was no difference between Hi and Lo genotypes in terms of all-cause mortality [RR=1.13 (95%CI 0.90-1.41;p=0.28)], recurrent myocardial infarction (95%CI 0.92-1.40; p=0.24) or frequency of coronary revascularization [RR=1.101 95% CI (0.78-1.57); p=0.56].

Conclusion

An association between 9p21-3 and greater baseline severity of coronary atherosclerosis was seen in this meta-analysis. However no association was seen between 9p21-3 and adverse angiographic and clinical outcomes which may be due to diminishing genetic risk following dietary modification and statin therapy.
Abstract 394: Metabolic Insights into Age and Gender: A New Algorithm for Regional Body Mass Distribution
Authors: Pownall Henry J , Rosales Corina , Gillard Baiba K , Gotto Antonio M
Published: July 2021

Metabolic syndrome (MetS), a disorder of energy metabolism associated with increased risk of cardiovascular disease (CVD), comprises a cluster of risk factors—low HDL-C, hypertension, hypertriglyceridemia, hyperglycemia and central (abdominal) obesity. Although other investigators noted that non central fat loss, especially femoral gluteal depots, are associated with CVD, the association was not as strong as that with central obesity. We hypothesized that age- and gender-dependent changes in body fat distribution would uncover an underlying cause of MetS. However, it is impossible to compare fat distribution based on anatomical circumferences (C) and skin folds (SF) because of the confounding effects of body mass index (BMI), which rises in the early decades and declines thereafter. We developed an algorithm that adjusts skin folds and circumference data for BMI to obtain a BMI-corrected adiposity index (BAI = C/BMI) for each depot. For comparison over time, we normalized the data set to the value in the third decade of life (BAI
3
= 100) and calculated a normalized BAI. as BAI
N
= [BAI
D
/BAI
3
] X 100. Application of this algorithm to the NHANES I data set of C and SF values, reveals BMI-independent changes in body fat distribution as a function of gender and age, with the most profound changes between the second and eighth decades of life occurring in waist C, which increases with time and buttock C, which decreases over the same time interval. In men the changes begin in the third decade of life. In women, the change does not occur until the beginning of the fifth decade of life, which is approximately the perimenopausal period. We propose a mechanistic model in which the underlying defect in MetS, is impaired fat storage in non-central depots, which results in the transfer of non-esterified fatty acids to skeletal muscle thereby impairing glucose disposal, to liver where it drives VLDL-TG synthesis and secretion, and to central fat depots giving the MetS waistline. The defect is not known but could be tissue-specific apoptosis or insulin resistance. If valid, this model would suggest that therapies that enhanced adipogenesis and/or insulin sensitivity in non-central depots could prevent or delay the onset of MetS.
Abstract 395: Lipoprotein Apheresis of Hypercholesterolemic Patients Mediates Vasoprotective Gene Expression in Human Endothelial Cells
Authors: Morawietz Henning , Goettsch Winfried , Brux Melanie , Reimann Manja , Bornstein Stefan R , Julius Ulrich , Ziemssen Tjalf
Published: July 2021

OBJECTIVE

Hypercholesterolemia is an important risk factor of cardiovascular diseases. Lipoprotein apheresis is an efficient strategy to reduce the serum low-density lipoprotein (LDL)-cholesterol and lipoprotein(a) levels and cardiovascular complications in patients with severe hypercholesterolemia. The underlying molecular mechanisms are not well-understood. In this study, we analyzed the impact of lipoprotein apheresis on gene expression in human endothelial cells.

METHODS

Human endothelial cells were stimulated with serum of hypercholesterolemic patients before and after lipoprotein apheresis. The expression of endothelial lipoprotein receptors, nitric oxide (NO) synthase and adhesion molecules was quantified by real-time PCR and Western blot.

RESULTS

Lipoprotein apheresis reduced the expression of the lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) in endothelial cells. Low-density lipoprotein (LDL) receptor expression remained unchanged. The mRNA expression of the endothelial nitric oxide synthase (eNOS) was increased with serum of hypercholesterolemic patients after lipoprotein apheresis. In contrast, endothelial expression of vascular cell adhesion molecule 1 (VCAM-1) was reduced in response to serum after lipoprotein apheresis.

CONCLUSION

Lipoprotein apheresis reduced the expression of the proatherosclerotic oxLDL receptor LOX-1 and adhesion molecule VCAM-1 and increased the expression of vasoprotective and NO generating eNOS in human endothelial cells in response to serum of hypercholesterolemic patients. These novel molecular mechanisms may account for the antiatherosclerotic and vasoprotective potential of lipoprotein apheresis in patients with hypercholesterolemia.
Abstract 403: Generation of PreBeta1 High Density Lipoprotein by CSL112 in Human Plasma
Authors: Diditchenko Svetlana , Gille Andreas , Wright Samuel D
Published: July 2021

The ability of apoA-I to remove cholesterol from atherosclerotic plaque is thought to underlie its inverse correlation with cardiovascular risk. CSL112 was designed as an infusible formulation of human apoA-I to rapidly remove plaque cholesterol and reduce risk after an ACS. We have recently shown that infusion of CSL112 into rabbits caused a strong and immediate increase in the ABCA1-dependent efflux capacity of plasma, an increase in plasma unesterified cholesterol and rapid subsequent esterification of the cholesterol. In the presence of human plasma, CSL112 was significantly more potent than human HDL
3
at enhancing cholesterol efflux, and the efflux elevation was predominantly via the ABCA1 transporter. Consistent with this observation, addition of CSL112 to plasma led to generation of high levels of PreBeta1-HDL, a favorable substrate for ABCA1. Phase 1 studies of CSL112 have confirmed the elevation of efflux capacity in humans and the rapid generation of PreBeta1. Here we examine the steps leading to formation of PreBeta1-HDL. Plasma spiked with 0.8 mg/mL CSL112 and held at 0[[Unable to Display Character: ⁰]]C did not increase PreBeta1-HDL levels. However, incubation at 37[[Unable to Display Character: ⁰]]C caused the generation of PreBeta1 that reached half-maximal levels at 15-20 min and maximal levels at 2 h (7-fold over baseline). By contrast, addition of HDL
3
to plasma caused minimal generation of PreBeta1-HDL at any time point. Native gradient gel electrophoresis showed that formation of PreBeta1 was accompanied by elevation in the size and protein content of HDL
3
and HDL
2
. These data suggest that the remarkable ability of CSL112 to elevate efflux and PreBeta1 derives from engaging plasma proteins in a process of lipoprotein remodeling.
Abstract 409: Size-dependent Acute Cellular Responses of Unbound Free Fatty Acids
Authors: Lee Eun-Soo , Lee Mi Kyoung , Kim Se-hwa
Published: July 2021

Introduction

The level of free fatty acids (FFAs) in serum determines the pathophysiological condition. Most FFAs are carried in albumin-bound forms, but very small portion is remained unbound. Due to the methodological limitations, there is little known about the effect of unbound form.

Objectives:

This study aimed to understand the effect of local increase of unbound FFAs.

Methods and Results:

We synthesized the nano- and micro-particles of linoleate (LNP and LMP respectively) in unbound forms. The sizes of linoleate particles in distilled water, in serum-free, and in serum-added culture media were measured by dynamic light scattering. We confirmed that the size of LNPs in serum-added media were ~ 200 nm in diameter without aggregation. By the treatments of LNPs and LMPs in macrophage cell line (RAW 264.7), we studied size-dependent cellular responses including viability, lipid storage, and intracellular translocation. Increased lipid droplets in LNPs-treated cells were analyzed based on label-free coherent anti-Stokes Raman scattering imaging. We also found that LNPs (250 μM), not LMPs, activated the translocation toward nucleus of fatty acid binding protein 4 (FABP4), a carrier of intracellular FFA, by western blotting and immunocytochemistry. It was noteworthy that the nano-sized unbound FFA could induce post-transcriptional gene expression unlikely micro-sized FFAs. In the cells whose FABP4 function was inhibited by the chemical inhibitor, the cell death was significantly increased through apoptosis in short exposed time. This cell death response was mediated by endoplasmic reticulum stress suggesting the protective role of FABP4 on the imbalanced FFA metabolisms.

Conclusions:

We showed the size-dependent cellular responses by nano- and micro-sized unbound FFAs and the protective role of FABP4 on the FFA metabolisms. These findings provide the insights into acute cellular responses of unbound FFA.
Abstract 411: Vasculoprotective Function of HDL from Cetp-deficient Subjects
Authors: Ossoli Alice F , Gomaraschi Monica , Pozzi Silvia , Nilsson Peter , Jessup Wendy , Kuivenhoven Jan Albert , Cefalù Angelo , Averna Maurizio , Sirtori Cesare R , Franceschini Guido , Calabresi Laura
Published: July 2021

Objective-

Cholesteryl ester transfer protein (CETP) is a plasma glycoprotein that catalyses the transfer of cholesteryl esters from HDL to the other plasma lipoproteins. Genetic deficiency of CETP is one of the known causes of primary hyperalphalipoproteinemia and represents a unique tool to evaluate how structural HDL alterations impact on HDL atheroprotective activity. Aim of the present study was to assess the vasculoprotective activity of HDL isolated from carriers of genetic CETP deficiency.

Subjects and Methods-

HDL and HDL subfractions were isolated from carriers of the R37X, Q165X and IVS7+1 CETP mutations and tested for their protein/lipid composition and their anti-inflammatory and NO-promoting activity in endothelial cells.

Results-

HDL and HDL3 from carriers proved to be as effective as control HDL in down-regulating cytokine-induced VCAM-1 expression and in enhancing eNOS expression in endothelial cells. Interestingly, carriers’ HDL2 were significantly more efficient than HDL2 from controls in inhibiting VCAM-1 and enhancing eNOS expression, likely due to their peculiar lipid composition and their higher content of apoE. On the contrary, carriers’ HDL and HDL subfractions were less effective than lipoproteins from controls in stimulating eNOS activation by phosphorylation and NO production, likely because of a reduced content of S1P in carriers’ HDL.

Conclusions-

Large, apoE-enriched HDL that accumulate in genetic CETP deficiency retain their ability to preserve endothelial cell homeostasis, supporting the use of pharmacological CETP inhibition to increase HDL levels and enhance HDL-mediated atheroprotection.
Abstract 442: Correlates of the Triglyceride/HDL Cholesterol Ratio: The Very Large Database of Lipids (VLDL-4)
Authors: Jones Steven R , Martin Seth S , Joshi Parag H , Elshazly Mohamed , Kulkarni Krishnaji R , Toth Peter P
Published: July 2021

Background

The Triglyceride/HDL Cholesterol Ratio (TG/HDL-C) is predictive of atherosclerosis and vascular disease risk. It is frequently elevated in the metabolic syndrome and diabetes and tracks with measures of insulin resistance.

Methods

We investigated the correlates of TG/HDL-C in 3,609 serial subjects of the 1,340,614 U.S. adults from the VLDL dataset who had apoB, hsCRP, serum insulin levels, hemoglobin A1C (A1C) measurements in addition to ultracentrifugation lipid measurement by VAP method (Atherotech, Birmingham, AL). A continuous LDL density parameter (LLDR) was calculated as the natural log of the ratio of dense to buoyant LDL-C subfractions. Remnant lipoprotein cholesterol (RLP-C) was defined as dense remnant VLDL3-C + IDL-C. TG/HDL-C was modeled using multiple linear regression.

Results

The strongest correlates of TG/HDL-C in model 1 were serum insulin, RLP-C, LLDR and Lp(a)-C with overall model r=0.801, adjusted r2=0.641, p<0.001. Model 2 substituted dense VLDL3-C and IDL-C individually as independent variables in lieu of their sum, RLP-C; overall model r=0.855, adjusted r2=0.730, p<0.001. IDL-C was inversely correlated while VLDL3-C was more strongly and positively associated with TG/HDL-C. A1C was not significantly correlated in the multivariate models (Table).

TG/HDL-C ratio represents a composite of metabolic and lipid atherosclerosis risk markers including insulin levels, LDL density, RLP-C, lp(a)-C, homocysteine, gender and apoB.
Abstract 443: ABCA1 Up-Regulation but Not CETP Inhibition is Predicted to Increase the Reverse Cholesterol Transport (RCT) Input Rate: a Simulation Study of HDL-C Raising Targets Using a Novel in-silico Model of Lipoprotein Metabolism and Kinetics
Authors: Lu James , Nanjee M. Nazeem , Brinton Eliot A , Hübner Katrin , Mazer Norman A
Published: July 2021

Introduction

HDL is believed to mediate RCT and hence lower CV risk. The recent failures of HDL-C raising drugs to improve CV outcomes raise questions on the link between HDL-C and RCT. We investigate the association between the two using an
in-silico
model.

Methods

The model describes the cyclic process of αHDL maturation from lipid-poor ApoA-I (pre-β1) and the regeneration of pre-β1 from αHDL particles via remodeling processes (e.g., particle fusion, SRB1 and CETP mediated lipid transfers). RCT is a complex process initiated by cholesterol efflux into plasma. In our model we assume that the ABCA1-dependent loading of cholesterol onto pre-β1 is the main contributor to cholesterol efflux and quantify this as the RCT input rate. The model is calibrated to literature data on normal and CETP deficient subjects and validated with data on ABCA1 deficiency. Using a virtual population of n=359 subjects with low HDL-C, we simulate two target modulations, 80% CETP inhibition and 100% ABCA1 up-regulation, chosen to raise HDL-C by comparable amounts.

Results

Table 1 shows that both target modulations increase HDL-C by approximately 50%. For ABCA1 up-regulation, this reflects an increased RCT input rate; while for CETP inhibition, the increase in HDL-C results from a decreased clearance rate. The model also predicts a larger increase in ApoA-I and a greater decrease in the pre-β1 level and fraction with ABCA1 up-regulation compared to CETP inhibition.

Conclusions

The level of HDL-CE reflects the ratio of the RCT input rate to the clearance rate. Our
in-silico
model provides a tool for evaluating HDL-C raising targets in terms of their differential effects on the underlying metabolic processes.
Abstract 445: Niacin Beneficially Alters the Secretion of Intestinal-derived TRL and HDL in a Rat Model of Insulin Resistance
Authors: Mangat Rabban , Borthwick Faye , Ullrich Tina , Jacome Miriam , Vine Donna F , Proctor Spencer D
Published: July 2021

Introduction

The intestine secretes discrete fractions of triglyceride (TG) rich apoB-chylomicrons (CM) and HDL particles into mesenteric lymph. Insulin resistance (IR) is associated with overproduction of intestinal CM however, the effect of IR on intestinal HDL secretion remains unknown. Niacin has been shown to reduce plasma TG while increasing HDL-C, however it is unknown if niacin modulates intestinal lymphatic CM and HDL secretion.

Objective

To determine the effect of niacin on the secretion and composition of intestinal lymphatic CM and HDL in a model of IR (JCR:LA-
cp
rat).

Methods

IR rats were fed control chow or chow supplemented with niacin (0.5% or 1% w/w) for 6 weeks. The mesenteric lymph duct was cannulated and lymph sampled following an intra-gastric intralipid (
fed
) infusion for 6hrs. The lymphatic CM (<1.006g/ml) and HDL (1.063-1.21g/ml) fractions were separated by density ultracentrifugation.

Results

IR rats over-secreted (2-fold) lymph apoB-CMs, while simultaneously reducing lymphatic apoA1-HDL (-47%) secretion compared to non-IR rats. In addition, CM and HDL particles from IR rats were found to be enriched in TG (64 and 86% respectively). Interestingly, niacin stimulated the secretion of lymph HDL (>60%), as well as attenuated lymph CM (-53%) secretion compared to control. Niacin treatment was also found to normalize the TG content of both lymph-CM and lymph-HDL particles.

Conclusion

IR appears to stimulate the intestine to over-secrete apoB-CM as well as reduce apoAI-HDL secretion into lymph, potentially contributing to particle dysfunction. Niacin may contribute to improving CVD risk by reducing the over-secretion of intestinal pro-atherogenic apoB-particles, whilst restoring the number and TG enrichment of intestinal anti-atherogenic apoAI-particles.
Abstract 463: TLT-1 Deficiency Affects Hypercholesterolemia and Progression of the Atherosclerotic Plaque in Apoe Null Mice
Authors: Gonzalez Marieli , Reyes Fiorella , Marrero Deborah , Washington A V
Published: July 2021

Platelet activation at sites of inflammation triggers the secretion of molecules that induce the transition of atherosclerosis from fatty streak to an acute disease, featuring an increased vulnerability of the atherosclerotic lesion that results in plaque rupture and thrombosis. TLT-1 (Triggering Receptor Expressed in Myeloid cells (TREM)-like transcript-1) is a molecule exclusively found in the α-granules of megakarocytes and platelets and has a demonstrated effect in inflammatory responses. Upon platelet activation, TLT-1 is moved to the platelet surface, while its soluble form, s-TLT-1, is secreted and detected in serum. Studies using the C57Bl/6

treml1
-

/-
mouse demonstrated a predisposition to hemorrhage after an acute inflammatory challenge suggesting that TLT-1 may be a key regulatory molecule in the interface between hemostatic and inflammatory mechanisms. Because we have found that sTLT-1 levels are significantly elevated in apoE mice when compared to wild type, we hypothesized that TLT-1 may be playing an important role in the progression of atherosclerosis. To address this possibility, we generated apoE

-

/-
/

treml1
-

/-
double knockout mice [DN]. Assessment of lesions after 4 weeks high-fat diet (HFD) demonstrated that at early stages, TLT-1 deficiency accelerates fatty streak formation. After 20 weeks on HFD, lesions in both apoE

-

/-
and [DN] mice progressed to an advance fibrous plaque stage. Although their lesion sizes were not substantially different, lesion compositions were. The mechanistic basis of these differences appears to be that the [DN] mice have significantly higher cholesterol levels when compared to apoE

-

/-
mice. The increased cholesterol levels extend to the

treml1
-/-

mouse when compared to wild type mice at 4 weeks on HFD, this difference, however, gradually subsides as wild type mice cholesterol levels increase over 20 weeks. Interestingly, cholesterol levels in 50 week old mice on chow diet revealed minimal differences between test and control mice suggesting the higher cholesterol levels are related to increased dietary intake. Our work defines a surprising role for TLT-1 in the regulation of serum cholesterol levels during atherogenesis.
Abstract 474: Atherosclerotic Macrophages Develop a Progressive Lysosomal Dysfunction that can be Rescued by Induction of a Lysosomal Biogenesis Program
Authors: Emanuel Roy , Sergin Ismail , Razani Babak
Published: July 2021

Recent reports of the proatherogenic phenotype of mice with a macrophage-specific autophagy deficiency has renewed interest in the role of the autophagy-lysosomal system in atherosclerosis. Lysosomes have the unique role of serving to process both exogenous material including the excess of atherogenic lipids and endogenous cargo including dysfunctional proteins and organelles. Surprisingly, little is known about the effect of an atherogenic environment on macrophage lysosomes. To address this, we utilize a variety of complementary techniques to show that oxidized LDL and cholesterol crystals, two of the commonly encountered lipid species in the atherosclerotic plaque, create a profound lysosomal dysfunction in cultured peritoneal macrophages. Disruptions in lysosomal pH, enzyme activity, proteolytic capacity, membrane integrity, and morphology are readily seen when cells are incubated with such lipids. Using flow cytometry to isolate resident tissue macrophages, we show that atherosclerotic plaque macrophages show features of dysfunctional lysosomes, a process that appears to be progressive with advanced plaque formation. These observations lead us to investigate whether stimulation of lysosomal function can ameliorate some of these effects. TFEB is the only known transcription factor that acts as a master regulator of lysosomal biogenesis, although its role in macrophages has not been studied. We show that overexpression of TFEB in cultured macrophages initiates a robust prodegradative response including induction of lysosomal genes and the generation of nascent lysosomes. Interestingly, this response can rescue several deleterious effects seen with atherogenic lipid loading including reductions in the secretion of the proinflammatory cytokine IL-1β and reductions in foam cell formation. Taken together, these data demonstrate that lysosomal function is markedly impaired in atherosclerosis and suggest that induction of a lysosomal biogenesis program can have anti-atherogenic effects.
Abstract 476: Shear Stress-induced Atherosclerotic Plaque Regression is Reversed by Regulation of Macrophage Mobility via Matrix Metalloproteinase Inhibition
Authors: Simeone Stefania , Ebrahimian Talin , Michaud Veronique , Lehoux Stephanie
Published: July 2021

Atherosclerotic plaques form in regions of low blood flow, whereas vessels exposed to high shear stress remain lesion-free. We hypothesized that exposing established atherosclerotic plaques to elevated shear stress leads to lesion regression by facilitating inflammatory cell movement within the plaque. We developed a model of arteriovenous fistula (AVF) in mice, where the right carotid artery is anastomosed into the jugular vein. LDLR-/- mice were placed on a high-fat diet. Control mice were sacrificed at week 12, which coincided with sham and AVF surgery. Sham and AVF mice were kept on a high-fat diet for a further 4 weeks. This procedure increases the shear stress in the brachiocephalic artery (BCA) and leads to a 51% plaque regression in AVF. All groups had comparable lipid levels. However, BCA plaque macrophage, smooth muscle cell and collagen content was halved in AVF. We observed greater gelatinase activity in plaques of AVF mice, suggesting a role for matrix metalloproteinases (MMPs) in plaque regression. MMP-9 and MMP-3 expression was increased in AVF plaques whereas MMP-2 and MMP-14 expression was decreased (p<0.05). A separate group of mice was therefore treated post-surgery with an MMP inhibitor, doxycycline, or with a TIMP-1 over-expressing plasmid. Both prevented the reduction in plaque size in the AVF group. To better define the mechanism of plaque regression in the AVF, we devised an endothelial cell (EC)-macrophage co-culture system where the ECs were exposed to high, low or no shear stress, and macrophages exposed to the EC effluent. There was a 2.5 fold increase in the migration of macrophages exposed to high shear effluent vs. low shear (p<0.05). This coincided with a 3-fold increase in the number of macrophages expressing activated β1 integrin in the high shear conditions. Uptake of apoptotic cells by macrophages was also 25% higher in the high shear vs. static (p<0.05). When repeated using the MMP inhibitor, GM6001, the high shear increase in migration was blocked in the presence of MMP inhibition; however, it had no effect on cell phagocytosis. Our findings suggest that shear stress acting on ECs may influence the cells within the plaque by increasing MMP activity allowing for better macrophage motility, an important feature of regressing plaques.
Abstract 497: Clinical Significance of Heparin Induced Thrombocytopenic Thrombotic Syndrome with Initiation of Anti-heparin Antibodies Negative: A Case Report and Literature Review
Authors: Shi, MD Qi , Paulino Robert , Kiel Michael , Wu Huimin , Samavedam Sandhya , Kakde Amit , Bo Jiang , Malik Qasim , Crevecoeur Louis , Ravi Vimal
Published: July 2021

Introduction

Heparin induced thrombocytopenia thrombosis Syndrome has a high mortality and morbidity in cardiac surgical patients in spite of early diagnosis and management. We present a case with multiple acute deep venous thrombosis (DVT) after using unfractionated heparin (UFH) during coronary artery bypass graft (CABG) with initiation of HIT antibodies negative.

Case presentation

A 70-year-old male complained of left leg pain 2 days after CABG for an acute ST-elevation myocardial infarction. Patient was treated with UFH intravenous during CABG. Platelet count was 78 x 109/L that was 130 x 109/L before the procedure. Anti-heparin antibodies IgM/IgG were negative. Venous Doppler ultrasound showed an acute deep venous thrombosis (DVT) in the left peroneal vein. However, after the patient was treated with Coumadin anticoagulation for 7 days with INR in therapeutic range (2-3), the patient presented for right low extremity pain. Platelet count was 40 x 109/L. Anti-heparin antibodies IgM and IgG were repeatedly positive. Venous Doppler ultrasound showed multiple bilateral lower extremities acute DVTs. Patient had thrombectomy and continued with coumadin anticoagulation at INR in therapeutic range for 3 months.

Discussion

HITTS is an immune-mediated drug reaction that occurs more frequently in patients undergoing cardiovascular surgery. Thrombosis caused by the HIT/T syndrome is most commonly venous thromboembolism. Thrombosis is arterial in about 20% of cases, including acute limb ischemia, myocardial infarction, or stroke. Previous clinical data has shown that about 40% of high titer-negative HIT antibodies were associated with a positive result in repeated testing several days later, which is the same as our presentation. The mortality and morbidity rates of HITTS are approximately 30% and 60%, respectively, and an additional ~10% of patients require amputations or suffer other major morbidity. However, with early recognition and intervention, mortality can be reduced from >30 to <10%. Obviously, repeated testing to identify patients who have HIT despite a negative initial HIT test in several days is greatly recommended. Diagnosis is essentially clinical and negative results from laboratory assays do not exclude the diagnosis.