bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 362: Acute Exposure to Air Pollution Aggravates Acute Myocardial Infarction and Subsequent Ischemic Heart Failure in Mice
Authors: Wolf Dennis , Marchini Timoteo , Anto Michel Nathaly , Dürschmied Daniel , Hilgendorf Ingo , Bode Christoph , Zirlik Andreas
Published: June 2021

Background:
Clinical, but not experimental evidence has suggested that exposure to air pollution particulate matter (PM) aggravates myocardial infarction (MI) in humans. Here, we aimed to describe mechanisms and consequences of an acute PM exposure in an experimental mouse model of MI.

Methods and Results:
C57BL/6J mice were exposed to an air pollution particulate matter (PM) surrogate (Residual Oil Fly Ash) by intranasal installation, prior to surgical permanent ligation of the left anterior descending coronary artery (LAD). Mice exposed to PM showed exaggerated ischemic heart failure with decreased fractional shortening and diastolic dilatation in echocardiography 6 month after MI. Histological analysis demonstrated an increase in the infarct area by 45 ± 12 % and enhanced inflammatory cell recruitment into the myocardium of PM-exposed mice 6 days after MI. Augmented cell recruitment was caused by increased activation of circulating myeloid and vascular endothelial cells. Consistently, PM exposure increased leukocyte recruitment a model of sterile peritonitis and in intravital microscopy. Mechanistically, PM exposure potentiated levels of circulating pro-inflammatory cytokines, such as of TNF-α by up to 327 ± 100 %. Increased activation of endothelial cells and leukocytes could be reversed by TNF-α antibody blockade. We identified alveolar macrophages as primary source of elevated cytokine production. Accordingly, specific
in vivo
depletion of lung macrophages by clodronate inhibited cytokine secretion, abolished leukocyte recruitment in intravital microscopy, and protected from cardiac inflammation after simultaneous PM exposure. Conversely, lymphocyte-free Rag1
-/-
mice where susceptible to PM, indicating that alveolar macrophages, but not lymphocytes, are the cause of the systemic inflammatory response following air pollution.

Conclusion:
Our data demonstrate that an acute exposure to environmental PM worsens MI and its clinical outcome in mice. These findings provide a novel functional link between air pollution and inflammatory pathways, and emphasize the importance of environmental factors in cardiovascular disease.
Abstract 363: Thrombin Activatable Fibrinolysis Inhibitor is a Regulator of Angiogenic Potential in Endothelial Cells
Authors: Bazzi Zainab A , Balun Jennifer L , Boffa Michael B
Published: June 2021

Angiogenesis, the sprouting of new blood vessels from existing vessels, is a process that is fundamental to both normal physiology, as well as disease processes such as atherosclerosis and cancer metastasis. A key regulator of the angiogenesis is the elaboration of pericellular proteolytic activity including plasmin and the matrix metalloproteases (MMPs). Thrombin activatable fibrinolysis inhibitor (TAFI) is a plasma zymogen that can be cleaved by thrombin, plasmin, or thrombin in complex with thrombomodulin (TM) to form activated TAFI (TAFIa). TAFIa cleaves carboxyl terminal lysine and arginine residues from substrates, including plasminogen-binding sites on cell surface receptors. TAFIa is thus a regulator of pericellular plasminogen activation. Plasmin regulates angiogenesis through MMP activation and degradation of the extracellular matrix (ECM). Previous studies have shown that TAFIa downregulates endothelial tube formation in a fibrin matrix, likely through inhibition of fibrin degradation. The current study was undertaken to evaluate if TAFIa is capable of modulating angiogenic responses of endothelial cells
in vitro
in the context of reconstituted basement membrane. We found that treatment of primary human umbilical vein endothelial cells (HUVECs) with a specific inhibitor of TAFIa, potato tuber carboxypeptidase inhibitor, resulted in an increase in proliferation, invasion, tube formation, and collagen degradation. On the other hand, treatment with a stable variant of TAFIa (TAFIa-CIIYQ) with a 150-fold increased half-life at 37°C resulted in a decrease in HUVEC tube formation and collagen degradation of HUVECs. Furthermore, a mutant soluble form of TM that supports TAFI, but not protein C, activation also had an inhibitory effect on HUVEC tube formation and collagen degradation. Finally, we assessed the effect of TAFIa on plasminogen activation. Treatment of HUVECs with various concentrations of TAFIa resulted in a decrease in uPA-mediated plasminogen activation on the surface of HUVECs. Together these experiments provide direct evidence of an anti-angiogenic effect of TAFIa. Our results suggest that promotion of TAFI activation or TAFIa stability may be viable therapeutic approaches for inhibition of angiogenesis.
Abstract 37: Measure of Appropriateness in the Placement of Intravenacaval Filters Among Guidelines From Major Medical Societies
Authors: Thyagarajan Braghadheeswar , Patel Shil , Swergold Natalie , Alagusundaramoorthy Sayee Sundar , Verma Isha , Baker Thomas , Eng Margaret
Published: June 2021

Objective:
With healthcare focusing towards quality based healthcare it has created significant interest among physicians in measuring and defining appropriateness in the treatment of medical conditions. We compared the indications for which the Intravenacaval (IVC) filter was placed in our hospital with the current guidelines by three of the major medical societies the
American Heart Association (AHA), American College of Chest Physicians (ACCP) and Society for Interventional Radiology (SIR)
to compare the measure of appropriateness in the placement of IVC filters among the guidelines

Methods:
We conducted a retrospective review of all charts with the ICD 9 code for placement of IVC filter from January, 2010 to January, 2015. Each patient chart was reviewed for patient demographics, complications and indications which were compared with the guidelines as defined by
AHA, ACCP and SIR

Results:
During the 5 years, 592 patients underwent the procedure which included 233 men and 359 women who had a mean age of 67.2 +/- 17.4. 75.8% filters were inserted by vascular surgery and 24.2% filters were inserted by interventional radiology. 1.8% of the patients had some form of complication either during insertion or retrieval. On comparing the indications, we found that 35.5%(AHA), 30.9%(ACCP), 35.5%(SIR) were appropriate. While 4.4%(AHA), 4.4%(ACCP), 47.4%(SIR) were relatively appropriate and 60.1%(AHA), 64.7%(ACCP), 17.1%(SIR) were not appropriate. Commonly used indications not defined clearly included prophylaxis for bariatric surgery (14.4%) and high risk for fall (11%). Statistical analysis with the Chi-square test showed there was no statistical difference between AHA and ACCP (p =0.243), while statistical difference was present between AHA and SIR (p <0.001) and ACCP and SIR (p < 0.001)

Conclusion:
This study demonstrates that there are significant differences between the guidelines from major medical societies for the placement of IVC filter which leads to compelling discrepancies amongst physicians in their decision for the placement of IVC filters
Abstract 376: Downregulation of Endogenous Apelinergic Axis Mediates Endothelial and Organismal Senescence
Authors: Rai Rahul , Ghosh Asish K , Smith Layton H , Vaughan Douglas E
Published: June 2021

Background:
Apelinergic signaling is a recently discovered GPCR mediated pathway. Endothelial cells are the main source of endogenous apelin (apln) while apelin receptor (aplnr) is present on multiple cell types. Since the role of endogenous apelinergic pathway within the context of senescence is largely unknown, we ask if levels of apln- aplnr vary with aging. We also investigate the effects of downregulated apln- aplnr on cellular and organismal aging.

Approach and Results:
To assess variations in endogenous apln- aplnr with aging, we compared their levels in 1 month (young) and 1 year old (old) WT mice. We noticed significant downregulation of apln- aplnr with chronological senescence in multiple tissues. Expression of apelin was also reduced with replicative senescence of endothelial cells. L-NAME administration, a model of stress induced senescence, also repressed aortic and cardiac apln. To address the mechanism involved in downregulation of apln- aplnr, we administered young wild type mice with Ang II. After a week of Ang II, there was significant downregulation of aortic apln and aplnr. Ang II and TGF-β also repressed apln and aplnr
in vitro
.

Next we investigated the effects of downregulated apln on endothelial cells. In response to shRNA mediated apelin knockdown, cells exhibited slower proliferation and upregulated senescence associated markers. We observed similar results when endothelial aplnr was blocked with an antagonist, ML221. In addition, apln and aplnr deficient mice also exhibited features of cardiovascular aging, including ventricular hypertrophy and lower EF. Importantly, aplnr deficient mice at eight months of age were also hypertensive.

Conclusion:
We provide a systematic assessment of senescence associated variation in levels of apln- aplnr. We demonstrate the role of Ang II- TGF-β axis in downregulating apln- aplnr during chronological and stress induced senescence
in vivo
and
in vitro
. We propose a novel model of Ang II- TGF-β induced senescence. Where in, with aging Ang II and TGF-β repress endogenous apln- aplnr. Downregulation of endogenous apln- aplnr axis decreases beneficial “youthful” effects of apelin, resulting in endothelial dysfunction and accelerated organismal aging.
Abstract 377: Phorbol 12-myristate 13-acetate Downregulates Tissue Factor Gene Expression in Human Pericytes
Authors: Sommerville Laura , Snyder Stacey , Hoffman Maureane
Published: June 2021

Tissue factor (TF) is a transmembrane receptor for factor VII/VIIa that plays a central role in hemostasis and angiogenesis. While TF is generally upregulated in pathologic conditions, we have reported that TF is downregulated in pericytes around vessels near a healing cutaneous wound. As the first demonstration of physiologic downregulation of TF, this finding suggested a potential means of modulating TF expression for therapeutic purposes. The goal of this study was to contribute to implementation of future therapies by elucidating mechanisms that regulate loss pericyte TF. To identify mediators of TF downregulation we utilized primary cultures of human placental pericytes to screen growth factors involved in wound healing including TGFβ, bFGF, VEGF, PDGF and ANG2. None significantly reduced pericyte TF expression as assessed by western blotting. We next tested agents that directly activate signaling pathways. Phorbol 12-myristate 13-acetate (PMA) triggered ~60% reduction in TF protein 4 hours after treatment. Complete loss of TF occurred by 8 hours (p<0.001) and remained until the experiment was terminated 24 hours after PMA addition. These results suggested that TF loss is mediated, at least in part, by downregulation of TF gene expression. We utilized qRT-PCR to determine the effects of PMA on synthesis of TF transcripts 4, 8, 12, and 24 hours after treatment. TF mRNA levels were unchanged 4 hours after treatment. However, TF transcripts decreased 4 and 6 fold 8 hours and 12 hours after PMA, respectively (p<0.01). 24 hours after treatment the amount of TF mRNA rebounded, yet remained 2 fold lower than the control (p<0.05). To determine if the decrease in TF transcripts is caused by mRNA destabilization, pericytes were treated with actinomycin D prior to PMA. Degradation of TF transcripts in PMA-treated cells was similar to that of DMSO-treated control cells at all time points (p=ns), indicating that PMA-mediated downregulation of TF expression occurs primarily through inhibition of mRNA synthesis rather than through destabilization of existing transcripts. In conclusion, our results demonstrate a role for transcriptional downregulation in PMA-mediated loss of pericyte TF protein, findings that pave the way for future
in vivo
studies of wound healing.
Abstract 38: Integration of Different Platelet Activity Assays Into a Reproducible Platelet Score
Authors: Berger Jeffrey S , Montenont Emilie , Karotkin Liza , Allen Nicole M , Nardi Michael A , Xu Jinfeng , Guo Yu , Hochman Judith S
Published: June 2021

Background:
While platelets play a key role in the pathogenesis of atherothrombosis, measuring platelet activity remains a research tool. We sought to 1) develop an integrated platelet score (PS) combining multiple platelet activity measurements, 2) assess its reproducibility over time, 3) investigate its correlation pre- and post-aspirin, and 4) compare subjects with cardiovascular disease (CVD) versus healthy controls.

Methods:
Following an overnight fast, 48 healthy controls had 4 weekly phlebotomies and received 81 mg aspirin daily between weeks 3 and 4. At each collection, platelet activity was measured using light transmission aggregation (LTA) in response to submaximal epinephrine, ADP, collagen, arachidonic acid (AA) and saline, and whole blood impedance aggregation in response to collagen, ADP and AA. The PS was derived using principal component analysis from healthy controls off antiplatelet therapy. A cohort of 124 subjects with CVD on aspirin was evaluated as a comparator group.

Results:
A PS was constructed using 12 measurements from LTA and whole blood aggregation, which accounted for 75% of the variation. The PS significantly correlated with all the platelet measurements used to construct the score (P<0.001 for each measurement). During weeks 1 to 3, the weekly correlation and reproducibility were excellent (r>0.77, ICC 0.82, P<.0001). After 1-week of aspirin therapy, the post-PS score shifted to lower values (P<0.001) and significantly correlated with the pre-PS score (r=0.64, p<.001). Among controls, female sex was the only variable associated with higher PS after multivariable adjustment (P=0.01). The PS was significantly higher in subjects with CVD (-123.8 [-151.5, -83.23]) versus healthy controls (-101.4 [-140.3, -62.03]; P=0.04).

Conclusion:
Platelet activity can be summarized into a PS that is reproducible over time, significantly associated with platelet activity measured on aspirin, and higher in women and subjects with CVD. Future studies are needed to investigate this score in other high-risk phenotypes and to determine its association with incident cardiovascular events.
Abstract 381: Chemogenetic Activation of Satellite Glial Gq-GPCR Signaling Enhances Cardiovascular Function
in vivo
Authors: Xie Alison X , Lee Jakovin J , McCarthy Ken D
Published: June 2021

The autonomic nervous system (ANS) exerts profound regulatory influence over cardiovascular physiology. The integrated network among autonomic nuclei in the central nervous system (CNS), as well as ganglionic innervation in the peripheral nervous system (PNS) are essential for homeostasis and for coordinating adaptive changes in physiology and behavior. In addition to neurons, the ANS contains large numbers of Glial Fibrillary Acidic Protein (GFAP) - expressing cells (GFAP
+
glial cells), which serve many functions in assisting neuronal network activity in both the CNS and the PNS. The present study explores the active role of GFAP
+
glial cells in regulating cardiovascular function
in vivo
. Using DREADD technology, we selectively activate Gq-GPCR signaling in satellite glial cells (SGCs) within sympathetic ganglia, which led to fast-acting, long-lasting increases in cardiac function and blood pressure
in vivo
. In addition, ganglionic SGCs likely increase postganglionic neuronal firing and subsequent norepinephrine release via a “local stimulatory effect“ on postganglionic neurons. Ongoing studies
ex vivo
using superior cervical ganglia cultures focus on identifying the mechanism underlying the interaction between sympathetic SGCs and postganglionic neurons.
Abstract 382: Cyclophilin A (CypA) is a Pathogenic Mediator of Pulmonary Arterial Hypertension
Authors: Xue Chao
Published: June 2021

Rationale:
Pulmonary arterial hypertension (PAH) is a devastating disease in which oxidative stress has been proposed to mediate pathological changes to the pulmonary vasculature such as endothelial cell (EC) apoptosis, endothelial to mesenchymal transition (EndMT), vascular smooth muscle cell (VSMC) proliferation, and inflammation. Our previous study showed that cyclophilin A (CypA) was secreted from EC and VSMC in response to oxidative stress, and much of the secreted CypA was acetylated (AcK-CypA). Furthermore, CypA was increased in the plasma of patients with PAH.

Objective:
To evaluate the cell-
s
pecific role of CypA in PAH and compare the relative effects of AcK-CypA and CypA on EC apoptosis, development of an inflammatory EC phenotype and EndMT.

Methods and Results:
Transgenic overexpression of CypA in EC, but not SMC, caused a PAH phenotype including increased pulmonary artery pressure, α-smooth muscle actin expression in small arteries, and CD45 positive cells in the lungs. Mechanistic analysis using cultured mouse lung microvascular EC showed that CypA and AcK-CypA increased apoptosis measured by caspase 3 cleavage and TUNEL staining. MM284, a specific inhibitor of extracellular CypA, prevented EC apoptosis. In addition, CypA and AcK-CypA promoted an EC inflammatory phenotype assessed by increased VCAM1 and ICAM1 expression, phosphorylation of p65, and degradation of IkB. Furthermore, CypA and AcK-CypA promoted EndMT assayed by change in cell morphology, increased mesenchymal markers and EndMT related transcription factors. At all concentrations, AcK-CypA stimulated greater increases in apoptosis, inflammation and EndMT than CypA.

Conclusions:
EC-derived CypA (especially AcK-CypA) causes PAH by a presumptive mechanism involving increased EC apoptosis, inflammation and EndMT. Our results suggest that inhibiting extracellular secreted CypA is a novel therapeutic approach for PAH.
Abstract 383: STIM1 Deficiency in Vascular Smooth Muscle Cells Promotes Vascular Calcification in Atherosclerosis
Authors: Yang Youfeng , Sun Yong , Chen Yabing
Published: June 2021

Vascular calcification is a characteristic feature of atherosclerosis. We and others have demonstrated that osteogenic differentiation of vascular smooth muscle cells (VSMC) contribute predominantly to the pathogenesis of vascular calcification in atherosclerosis. The key cellular event that leads to calcification is the secretion of matrix vesicles (MVs). However, the molecular regulation of MV release and the causal effect of MV release on VSMC calcification are poorly understood. The objective of this study is to investigate the function of a key calcium flux regulator, stromal interaction molecule 1 (STIM1), in regulating MV release and VSMC calcification; and to elucidate the underlying molecular mechanisms. SMC-specific STIM1-deficient mice (STIM1
Δ/Δ SMC
) were generated by breeding SM22α-Cre mice with STIM1 smooth muscle floxed mice (STIM1
f/f
). In vitro characterization using primary VSMC isolated from STIM1
Δ/Δ SMC
and the control STIM1
f/f
mice demonstrated that STIM1 deletion promoted VSMC calcification, although STIM1 deficiency has been linked to decreased calcium signals in smooth muscle cells. Increased release of MVs was demonstrated with the STIM1
Δ/Δ SMC
VSMC compared with the control STIM1
f/f
VSMC. Using the atherogenic ApoE
-/-
model, we demonstrated that SMC-specific STIM1 deficiency increased atherosclerotic vascular calcification in vivo. Consistently, increased MVs were determined in the serum of the STIM1
Δ/Δ SMC
mice. Mechanistically, we found that STIM1 deficiency did not affect VSMC proliferation and survival, but decreased the expression of SMC-specific α-actin. The size of MVs released from STIM1
f/f
and STIM1
Δ/Δ SMC
VSMC appeared similar. However, MVs from the STIM1
Δ/Δ SMC
VSMC contained a greater amount of calcium compared with those from the STIM1
f/f
VSMC. Furthermore, immunofluorescent staining identified a rearrangement of actin-filament structure in the STIM1
Δ/Δ
VSMC, a critical cellular event that controls the release of MVs. These studies have determined a new causative effect of VSMC-expressed STIM1 on atherosclerotic vascular calcification; and identified a novel link connecting STIM1 and actin-cytoskeleton rearrangement in regulating MV release and vascular calcification.
Abstract 39: Platelet Reactivity to Prostaglandin E2 is a Novel Modifiable Risk Factor for St-Elevation Myocardial Infarction
Authors: Friedman Eitan A , Haddad Elias V , Joksas Valentinas , Huang Shi , Xu Meng , Boutaud Olivier
Published: June 2021

Background:
Patients with lower thresholds for platelet activation are at increased risk for primary and recurrent myocardial infarction (MI) and overall cardiovascular (CV) mortality. We have demonstrated that there are two phenotypes of platelet response to Prostaglandin E
2
(PGE
2
), such that it increases threshold for aggregation in 45% of individuals (inhibitory) and lowers threshold for aggregation in 55% (potentiating). As PGE
2
is present in atherosclerotic plaques, and its receptors are present on platelets, biologic variability in PGE
2
responses may have clinical implications. We hypothesized that patients with higher thresholds for platelet activation would have a lower risk of thrombotic CV events, specifically ST-Elevation MI (STEMI).

Methods:
85 patients undergoing percutaneous coronary intervention for stable or unstable coronary disease were phenotyped for PGE
2
response. Platelet rich plasma was treated with various concentrations of U46,619 (thromboxane agonist) with or without PGE
2
100 nM, and phenotype determined by light aggregometry. Analysis of the maximum PGE
2
effect (maximum aggregation with PGE
2
minus maximum aggregation without it) was performed using linear and non-linear statistical methods.

Results:
Traditional cardiovascular risk factors were similar between groups. A higher percentage of patients with the potentiating phenotype had a history of STEMI than those with the inhibitory phenotype. Logistic regression using restricted cubic spline showed that the predicted probabilities of STEMI increased from 0.04 (at the strongest inhibitory phenotype) to 0.43 (at the median phenotype). The OR of phenotype at the median relative to that at the 10th quantile was 7.4 (95 % CI=1.6, 34.8).

Conclusions:
PGE
2
inhibitory phenotype confers a decreased lifetime risk of STEMI in individuals at high risk for CV events. We have previously shown that an EP3 receptor antagonist converts the potentiating to the inhibitory phenotype. Thus, the PGE2 phenotype may be a novel marker of cardiovascular risk that may also identify patients who would benefit from an EP3 antagonist.
Abstract 393: Covalently Connected Apolipoprotein A-I Molecules Provide Support for the Double Belt Model in Reconstituted HDL
Authors: Morris Jamie C , Davidson W. Sean
Published: June 2021

Because of its critical role in high density lipoprotein (HDL) metabolism, significant work has been devoted to studying apolipoprotein (apo)A-I structure in its lipid-bound state, particularly reconstituted forms of HDL (rHDL). Unfortunately, high-resolution structural studies have not been successful because of HDL heterogeneity and compositional dynamics. There is general agreement that apoA-I adopts an antiparallel arrangement on rHDL with the majority of the molecule conforming to the “double belt” model. However, less is understood about the locations of the N- and C-termini in these particles. The classic double belt predicts that, in a particle containing two molecules of apoA-I, all four termini are localized to one area of the particle. However, other models predict that they are far apart. To address this issue, we generated a series of covalently connected apoA-I dimers and tested their ability to generate rHDL particles. The dimeric (d)-apoA-I
C-N
mutant, locked the C-terminus of one apoA-I molecule to the N-terminus of a second molecule with three intervening Ala residues. Linked by strategically introduced cysteine residues, the d-apoA-I
C-C
, and d-apoA-I
N-N
mutants lock the C-termini and the N-termini of two apoA-I molecules together, respectively. When all three of these mutants were reconstituted into rHDL particles using sodium cholate and synthetic phospholipids, the resulting particles closely resembled those generated with wild-type (WT) apoA-I. This indicates a ring-like structure predicted by the double belt. However, when testing the ability of these mutants to spontaneously clear lipid, we found that both the N- and C- termini required the ability to separate and move independently to form normal particles. Native gel analysis showed the dimerized lipid-free mutants present as a tight homogenous band compared to wild-type which runs as a mixture of oligomers. We are currently evaluating if the homogeneity of these mutants can allow for more facile crystallization of full-length apoA-I. Further studies with these stabilized mutants should help us understand the structural basis underlying HDL’s diverse functions, which is essential for developing HDL-based therapies for cardiovascular disease.
Abstract 394: ApoE3-containing High Density Lipoprotein Serves as a Multifunctional Platform for Delivery of Gold Nanoparticles for Hyperthermic Treatment
Authors: Narayanaswami Vasanthy , Chuang Skylar T , Shon Young-Seok
Published: June 2021

We have developed a novel HDL-based multifunctional platform for transport and delivery of highly hydrophobic gold nanoparticles (AuNP) bearing photothermic properties. We exploit the ability of apolipoprotein E3 (apoE3) to act as a high affinity ligand for the low-density lipoprotein receptor (LDLr) to gain entry into glioblastoma cells. The issue of poor aqueous solubility of AuNP was overcome by integrating them with phospholipids and apoE3, yielding reconstituted rHDL bearing 3, 10, or 10 nm AuNP. UV-Vis spectra of rHDL-AuNP indicated the presence of stable particles with surface plasmon band at ~530 nm, a signature feature of AuNP. Transmission electron microscopy (TEM) revealed discoidal geometry for rHDL with 3, 10 and 17 nm particles. Co-immunoprecipitation assay using a soluble form of the LDLr indicated robust binding of rHDL-AuNP to the receptor. Immunofluorescence analysis indicated that all 3 rHDL particles were internalized by glioblastoma cells, as revealed by the presence of punctate, peri-nuclear endocytic/lysosomal vesicles; this suggests cellular uptake of rHDL-AuNP by receptor-mediated endocytosis. Cellular uptake was further confirmed by TEM, in which aggregated AuNP were found in the endosomal-lysosomal compartments. Finally, cytotoxicity studies demonstrated that more than 50% of cells were viable with rHDL-AuNP treatment of up to 0.1 mg/ml for 24 h. The findings that apoE3: (i) acts as a detergent in solubilizing and dramatically improving the aqueous solubility of AuNP, and, (ii) facilitates cellular uptake of rHDL-AuNP by the LDLr pathway, are significant since they offer an effective means of delivering AuNP across the cell membrane. This is particularly relevant in tumor cells since they overexpress LDLr to meet the high demand for cholesterol that is required for rapid proliferation and membrane biogenesis.
Abstract 396: Diet-derived Lysophosphatidic Acid and Obesity-associated Cardiovascular Disease Risk
Authors: Onono Fredrick , Mueller Paul , Brandon J. , Soman Sony , Sunkara Manjula , Smyth Susan , Morris Andrew
Published: June 2021

Diet-dependent obesity is a risk factor for coronary artery disease and a powerful risk factor for some cancers particularly in women. One mechanism that has been proposed to link diet-dependent obesity to disease is that diets high in fats and lipids promote the formation of bioactive lipid metabolites that increase disease risk. Lysophosphatidic acid (LPA) is a family of bioactive lysophospholipids that elicit broadly mitogenic and proinflammatory responses in multiple cell types, primarily through actions at cell surface G-protein coupled receptors. LPA promotes vascular smooth muscle cell differentiation and migration, decreases vascular barrier endothelial function, activates platelets and myeloid cells all of which could contribute to the development of atherosclerosis. In mice, genetic and pharmacological targeting of LPA receptors inhibits atherosclerosis. We conducted studies to investigate the impact of fasting and high fat feeding on circulating levels, metabolic sources and distribution of plasma LPA in both humans and mice. After overnight fasting, blood was drawn from human volunteers to obtain baseline LPA levels before being given a drink supplemented with fats to provide approximately 50% of daily energy requirement. Blood was subsequently drawn at intervals to prepare plasma. In mice, after overnight fasting, the animals were gavaged with olive oil either with or without a tracer phosphatidylcholine. These studies were extended to mice with genetically induced hyperlipidemia fed models of diet. Changes in plasma LPA levels were measured by using HPLC ESI multistage mass spectrometry. We found that plasma LPA levels in mice and humans are acutely sensitive to fasting and high fat feeding. In mice plasma LPA levels are dramatically increased by a combination of feeding a high fat atherogenic diet and genetic induction of hyperlipidemia by inactivation of the LDLr or apoE genes. In addition, in mice, plasma LPA can be formed from dietary phosphatidylcholine. These results provide support for the concept that high fat atherogenic diets promote the formation of circulating LPA and have led to development and characterization of mouse models that can be used to define the role of LPA in obesity in other disease processes including cancer.
Abstract 404: A High Dose Olive Oil, Polyphenol, and Lectin Limited Diet Reverses and/or Stabilizes Advanced Coronary Artery Disease
Authors: Gundry Steven R , Epstein Jean
Published: June 2021

Introduction:
Coronary Artery Disease (CAD) is thought to be progressive; standard treatment protocols call for instituting a low fat/low cholesterol diet program, exercise, and lipid lowering agents. This results in an approximate 30-40% new event rate in 5 yrs. We evaluated our treatment strategy to reverse CAD with The Corus Score (CS) (Cardiodx, Redwood City, Ca), proven to quantify coronary artery obstructive plaque by the expression of 23 genes.

Methods:
Based upon using a Lectin-limited diet to prevent/reverse Metabolic Syndrome and CAD, we have enrolled and followed 800 pts (aged 42-89 yrs) with known CAD, defined as previous MI, stent, CABG, or positive stress test/angiogram, positive CS greater than 30, into a physician coached program, which reduces grains, legumes, nightshades, seeded vegetables, Casein A1 milk, (the all lectin containing food groups),and fruits; emphases consumption a liter of olive oil/wk, large amts of green vegetables, and 4 oz amts of proteins, avoiding commercial poultry (Matrix Protocol). All Apo E 4 genotypes avoided animal fats and cheeses. Pts were instructed to take 4,000 mg of high DHA fish oil, 200mg of Grape Seed Extract, and 25 mg of Pycnogenol per day, and consume polyphenol rich coffee and/or teas and 1 oz dark chocolate/day. Diets/supplements were individualized based on results of Advanced Cardiovascular Risk Markers (ACRM), which were sent to two core labs. Yearly assessment of CAD severity was measured by Corus Score (possible range 1-40). Any score above 30 was assessed by Nuclear Stress testing.

Results:
Pts have been followed for 1.5 to 6 years (mean 4.5 yrs). Only 6/800 pts (0.5%) have received a new stent, all 6 had rising Corus scores: two also had a rising Lp-PLA2, 2 had rising Cardiac Troponin I levels; one pt required CABG: . There have been no MI’s, unstable angina. Corus scores at baseline decreased from 34+/-4 (range 6-36) to 24+/-3, P<0.01. Only 64/800 pts (8%) had a rise in Corus scores/ 736/800 pts’ CS declined or remained stable (92%). Only 6/64 Corus scores had positive Stress tests.

Conclusions:
Simple Nutrigenomic-based dietary interventions, based upon ACRM's and Corus Scores, represents a quantum leap forward in preventing/modifying Cardiovascular events in known CAD patients.
Abstract 405: Utility of Very Low-density Lipoprotein Cholesterol to Total Triglyceride Ratio (VLDL-C/TG) and Non-VLDL TG to Quantify Remnant Lipoprotein Cholesterol (RLP-C): The Very Large Database of Lipids Study 7C
Authors: Hendrani Aditya D , Quispe Renato , Martin Seth S , Kulkarni Krishnaji R , Toth Peter P , Joshi Parag H , Jones Steven R
Published: June 2021

Background:
RLP-C is comprised of atherogenic triglyceride- (TG-) rich lipoproteins, commonly defined as the sum of intermediate-density lipoprotein cholesterol (IDL-C) and very low-density lipoprotein cholesterol remnants (VLDL
3
-C). In clinical practice, the VLDL-C/TG ratio is used to diagnose type III dyslipidemia, a primary lipoprotein disorder characterized by high levels of RLP-C.

Methods:
Serum lipids of 556,307 U.S. adults with TG ≥130 mg/dL were analyzed by ultracentrifugation (VAP, Atherotech, Birmingham, AL). We estimated TG content in VLDL (VLDL-TG) as the product of VLDL-C and validated variable TG/VLDL-C factors. Non-VLDL-TG was then calculated as total TG minus VLDL-TG, for which negative values represented the presence of RLP-C. We examined the relationship of non-VLDL-TG to 1000 quantiles of VLDL-C/TG ratio. We defined a VLDL-C/TG ratio cutpoint for presence of RLP-C based on the quantile at which median non-VLDL-TG≤0.

Results:
We found median non-VLDL-TG≤0 at VLDL-C/TG = 0.18
(Figure)
. There were 174,907 adults who did not meet diagnostic criteria for type III dyslipidemia (VLDL-C/TG 0.18 to <0.30), whose levels of RLP-C and non-VLDL-TG levels were 37 (31-46) and -20 (-40 to -8) mg/dL, respectively. A total of 1,550 adults met classical diagnostic criteria for type III dyslipidemia (VLDL-C/TG ≥0.3), whose plasma levels of RLP-C and non-VLDL-TG levels were 80 (67-101) and -187 (-290 to -129) mg/dL, respectively.

Conclusion:
A threshold of VLDL-C/TG ≥0.18 correlates with the accumulation of RLP-C in plasma. If validated in future studies, these findings will improve identification of individuals who are at greater risk for atherosclerotic disease.
Abstract 408: Endothelin-1 and Vitamin D
3
Potentiated Calcification and Outward Vascular Remodeling in Leptin-deficient
ob/ob
Mice
Authors: Carmo Luciana S , Almeida Youri E , Andrade Maria C , Silva Elisangela F , Liberman Marcel
Published: June 2021

Vascular remodeling, which is an adaptive response of the vessel to specific stimuli has a pivotal role in cardiovascular disease, such as aneurysm formation. In that scope, serum endothelin-1 (ET-1) which is increased in diabetic patients, may contribute to atherosclerosis and aneurysm development. We hypothesized that leptin-deficient ob/ob mice may demonstrate an exaggerated vascular remodeling associated with vascular calcification after Vitamin D
3
in vivo
. We further investigated the role of ET-1 in vascular smooth muscle cells (SMC) osteochondrogenic differentiation and calcification
in vitro
. We used leptin-deficient (obob, n=12) and C57BL/6 mice (C57, n=12) injected with saline (Cont) or Vitamin D
3
8x10
4
IU (VitD) i.p. daily for 14 days. Results are shown as Mean±SEM and considered statistically significant if p≤.05 by ANOVA. Aorta from ob/obVitD increased both external and internal elastic lamina circumferential length (3478±269.7μm and 3186±262.6μm) versus (vs.) ob/obCont (2841±74.73μm and 2657±71.37μm) vs. C57VitD (2611±39.62μm and 2333±41.79μm) and vs. C57Cont (2569±164.6μm and 2351±167.7μm), thus characterizing outward vascular remodeling. We found that vascular calcification area strongly correlated with increased total vessel area (r
2
=0.8, p=0.003). We also incubated primary SMC isolated either from obob or from C57 aorta without (Cont) or with ET-1 50nM (ET) for 0-72h. ObobET SMC increased calcification vs. obobCont (1,69±0.025 vs. 1.03±0.03) after 14 days p<.05, n=3 and C57ET did not calcify (0.97±0.02 vs. C57Cont 1±0.05), p=NS. Concomitantly, obobET SMC increased osteochondrogenic differentiation, by modulating RUNX2 (1.23±0.03 vs. obobCont 1±0.01, p<.05 n=3) after 48h, which did not occur in C57. Furthermore, ERk1/2 dephosphorylation peaked after 30min in C57ET in comparison to 60min in obobET SMC. Concurrently, VitD increased both ETA and ETB receptor (4.00±1.04 and 4.25±1.25) in obob aorta vs. paired control
in vivo
. Nevertheless, C57VitD increased ETA receptor mRNA expression (5.44±1.08) only. In conclusion, we uncovered signaling pathways that may connect Vitamin D and ET-1 calcifying effect to induce excessive outward remodeling in type 2 diabetes.
Abstract 409: Notch Signaling in Bone Marrow-derived FSP-1
+
Cells Mediates a Phenotypic Change in Smooth Muscle Cells Leading to AVF Failure
Authors: Cheng Jizhong , Liang Ming , Luo Jinlong , Du Jie
Published: June 2021

Background:
sociological and economic costs of failed arteriovenous fistulas (AVF) are well known but mechanisms of AVF losses are controversial. We have shown that smooth muscle cells (SMCs) from anastomosed artery compose of ~50% of neointima cells in AVF. Arterial anastomoses are also infiltrated by bone marrow (BM)-derived FSP-1
+
cells, potentially linking FSP-1
+
cells to SMC activation. However, whether Notch/RBP-JK signaling involves in BM-derived FSP-1
+
cells activation has not been studied. We propose that Notch/RBP-JK activation in BM-derived FSP-1
+
cells causes a phenotype-switch of SMCs, resulting in SMC translocation from the artery to the venous anastomosis, forming neointima.

Methods:
we created CKD and AVFs in wild type and Notch knock out mice. The role of Notch signaling in activation and function of BM-derived FSP-1
+
cells was explored. Activated SMCs were characterized by a loss of differentiation markers plus a gain in proliferation (PCNA+). Using BM transplantation, we examined if FSP-1
+
cells lacking RBP-JK in CKD mice would suppress SMC phenotype switch and neointima formation in AVFs.

Results:
BM-derived FSP-1
+
inflammatory cells in AVFs from CKD mice were 45% greater vs. results in pair-fed, control mice. In the artery anastomosis, increased infiltration of FSP-1
+
cells led to loss of SMC differentiation markers, SMMHC, SM-22 and SMA-α. Activation of Notch signaling (N1ICD
+
and RBP-JK
+
) raised expression of cytokines (IL-1β, MCP-1) and growth factors (PDGF-BB, bFGF2 and TGF-β1) in FSP-1
+
cells, The cytokines and growth factors caused a SMC phenotype switch (characterized by loss of SMC markers), resulting in SMC migration and proliferation. RBP-JK KO in

BM-derived FSP-1
+
cells significantly decreased the production of cytokines and growth factors, suppressed SMC activation of and neointima formation in AVFs.

Conclusions:
Notch signaling in BM-derived FSP-1
+
cells can induce a phenotypic switch in SMCs stimulating neointima growth. Targeting Notch signaling in FSP-1
+
cells could improve AFV function.
Abstract 411: Obesity Alters the Expression of Very Low Density Lipoprotein Receptor (vldlr) Expression in Peripheral Blood Monocytes: Possible Links Between Vldlr Expression and Monocyte Phenotypes
Authors: Hajri Tahar , Johnson Brian , Mazpule George , Talishinskiy Toghrul , Eid Sebastian , Ewing Douglas , Novack Richard , Novack Richard , Schmidt Hans ,   Bariatric Surgery Group
Published: June 2021

Objective:
Very low density lipoprotein receptor (VLDLR) is a member of the low-density lipoprotein receptor family that binds multiple ligands including TG-rich lipoproteins. The role of VLDLR in lipid uptake had been demonstrated in cell culture and animal models, but little is known about the effects of obesity on VLDLR expression in human. Accordingly, we examined the impact of obesity on VLDLR expression in peripheral blood monocytes and explored possible links with monocyte inflammatory markers and circulating lipids.

Subjects and measurements:
Blood was collected from a total of 69 subjects including 23 normal-weight (body mass index: BMI < 25 kg/m
2
) controls and 46 obese (BMI > 30 kg/m
2
) subjects. Blood lipids, glucose and hormones were measured. Monocytes were isolated from buffy coats using Ficoll density centrifugation followed by magnetic separation of CD14-positive cells with magnetic beads. Expression of VLDLR, and pro- and ant-inflammatory markers of monocytes was examined by western blotting and quantitative qPCR. The uptake of labeled VLDL (DiI-VLDL) was also assessed in monocyte cultures.

Results:
Insulin, and total and VLDL triglycerides were increased in plasma of obese subjects. In addition, VLDLR protein and mRNA levels were significantly (p< 0.05) higher (~ 40%) in obese compared to lean subjects. Monocytes of obese subjects exhibited higher expression of IL-6 and TNF-α (+29 to 43%), but lower expression of IL-10 and CD163 (-30 to 47%). The levels of monocyte VLDLR was positively correlated with blood triglycerides and insulin, and negatively with adiponectin. The uptake of labeled VLDL, assessed by fluorescence microscope, was higher in monocytes of obese subjects.

Conclusions:
Our findings indicate that obesity upregulates the expression of VLDLR and pro-inflammatory markers in peripheral blood monocytes. The uptake VLDL was also increased in monocyte of obese subjects suggesting that obesity-induced VLDLR expression may explain the changes of VLDL uptake and monocyte phenotype.
Abstract 420: G3BP1 Activation of NFATc Signaling is Inhibited by LRP6 in Aortic Vascular Smooth Muscle Cells
Authors: Ramachandran Bindu , Stabley John , Behrmann Abraham , Cheng Su-Li , Mead Megan , Williams Bart , Towler Dwight A
Published: June 2021

When fed a high fat diet, male LDL receptor -deficient (LDLR-/-) mice develop type 2 diabetes (T2D) with concomitant arteriosclerotic calcification. We recently identified that vascular smooth muscle (VSM) LRP6 expression inhibits pro-sclerotic noncanonical Wnt signaling, mediated in part by protein arginine (Arg) methylation relays that converge on NFAT- dependent transcriptional programs. To better understand the mechanisms whereby Arg methylation mediates LRP6 actions,immunoaffinity mass spectrometry analysis was performed on aortic VSM extracts from SM22-LRP6(fl/fl);LDLR-/- and LRP6(fl/fl);LDLR-/- control mice. Applying a 2.5- fold cutoff threshold, alterations were noted in 499 monomethylated (MMA), 109 symmetric dimethylated (SDMA), and 65 asymmetric dimethylated (ADMA) protein cognates. One protein, G3BP1 (GTPase activating-protein binding-protein 1), demonstrated increases in MMA on residues 433, 435 & 458 -- with concomitant reductions SDMA on residue 458 —with LRP6 deficiency. G3BP1 exhibited both nuclear and non-nuclear localization, consistent with NTF2 and RRM structural motifs. Transient transfection and MMA western blot analysis confirmed that protein Arg methyltransferase 1 recognized G3BP1 as a substrate. This modification was inhibited by co-expression of LRP6. G3BP1activated osteopontin (OPN)- and NFATc3- dependent transcriptional responses, with NFAT transcription enhanced by co-expression of either Fzd10 or constitutively active cdc42. LRP6 inhibited G3BP1+NFAT activation of artificial and native (OPN, TNF) NFATc3-targeted promoter - reporter constructs. Moreover, NFATc3 protein levels were increased in nuclear extracts of LRP6-deficient VSM and reduced by G3BP1-directed siRNA, with concomitant reduction in genomic targets. Structure-function analysis mapped transcriptional activation of NFAT signaling by G3BP1 to the 32 amino-acid C-terminal domain encompassing Arg methylation cognates. Thus, G3BP1 is a cellular target of Arg methylation that is negatively regulated by LRP6 and mediates inflammatory osteogenic signaling in VSM. Strategies targeting G3BP1 and/or its Arg-methylation may help limit arteriosclerotic calcification and vascular disease in the setting of type 2 diabetes.
Abstract 425: Brown Fat Activation Enhances the Lipid-lowering and Anti-atherogenic Effect of Statin Treatment
Authors: Hoeke Geerte , van Dam Andrea D , Boon Mariëtte R , Berbée Jimmy F , Rensen Patrick C
Published: June 2021

Introduction:
Brown adipose tissue (BAT) produces heat by burning triglyceride (TG)-derived fatty acids. As a result, BAT accelerates the formation and hepatic clearance of cholesterol-enriched lipoprotein remnants via the apoE-LDL receptor (LDLR) pathway, thereby alleviating hypercholesterolemia. Since statins upregulate hepatic LDLR expression, the aim of this study was to investigate whether BAT activation and statin treatment cooperate in lowering hyperlipidemia and atherosclerosis.

Methods and Results:
APOE*3-Leiden.CETP
mice were fed a Western-type diet and treated with the selective β3-AR agonist CL316243 that activates BAT (CL, 20 μg/day; s.c.), atorvastatin (statin, 0.0036% w/w, in diet) or both. BAT activation alone and combined with statin treatment increased energy expenditure (+15%) as a result of increased fat oxidation (+50%). Furthermore, BAT activation decreased the lipid droplet content within BAT (-50%) and induced browning of subcutaneous white adipose tissue. BAT activation alone and combined with statin treatment also lowered plasma TG levels (-60%). In addition, plasma cholesterol was lowered by both BAT activation (-29%) and statin (-31%), but even further by the combination (-44%). The uptake of TG-derived fatty acids from glycerol tri[3H]oleate and [14C]cholesteryl oleate-labeled VLDL-mimicking particles into BAT was enhanced by BAT activation (+234%) and on top of statin (+220%), but not by statin alone. The hepatic uptake of the cholesterol-enriched remnants tended to be increased by BAT activation (+18%) or statin (+22%), and was markedly increased by the combination of BAT activation and statin treatment (+70%). Hepatic cholesterol levels were reduced by statin alone and the combination (-41%). Importantly, atherosclerosis development was decreased by BAT activation (-42%) and statin (-50%) alone and further decreased by the combination (-70%).

Conclusions:
BAT activation enhances the lipid-lowering and anti-atherogenic effect of statin treatment. We postulate that combining statin treatment with BAT activation is a promising new avenue to combat hyperlipidemia and cardiovascular diseases.