bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 210: Liver Expression of Proinflammatory Cytokines in Adiponectin Deficient Mice
Authors: Jha Arshi , Yu Esther , Pamidimukkala Jayabala
Published: June 2021

Adiponectin is one of the few peptides secreted by fat known to have anti-inflammatory properties. Obesity in humans is associated with low levels of circulating adiponectin. Adiponectin deficiency could potentially lead to a proinflammatory milieu, increasing susceptibility to progression of cardiovascular diseases and other inflammatory conditions. Previous studies from our lab have shown that adiponectin deficiency in mice did not lead to statistically significant increases in circulating levels of proinflammatory cytokines under basal conditions or in response to low dose infusion of Angiotensin II (ANGII). The present study evaluates the effect of chronic ANGII infusion on expression of cytokines in the livers of female C57BL/6J and adiponectin deficient mice (Adipo -/-). Female mice (24-28wks), were implanted with osmotic pump containing either ANG II (800 ng/Kg /min) or saline. Blood samples and tissue were collected at the end of 14 days. Liver tissue expression of the cytokines were quantified using real-time PCR (BioRad CFX 96 thermocycler) and SYBR Green mastermix. Data was normalized to the expression of GAPDH. Expression of cytokines IL6, IL8, IL17a was undetectable in the liver tissue of C57BL/6J and Adiponectin deficient mice. Angiotensin II infusion did not produce detectable changes in cytokine expression in either group. Expression of TNFα, TGF
β
, and adhesion molecules such as E-Selectin, VCAM1, Collagen1a1 in Adipo-/- mice were comparable to that of C57BL/6J mice. ANGII infusion did not produce statistically significant changes in these biomarkers either. Finally, expression of ANGII signaling components such as Angiotensin type 1 receptor (AT
1
), NFκB and PPARγ were robustly expressed in both C57BL/6J and adiponectin deficient mice. In conclusion, chronic infusion of ANGII in Adiponectin deficiency did not result in a significant increases in expression of markers of proinflammatory milieu in the liver tissue.
Abstract 213: Differential Effects of Nanoformulated Copper/zinc Superoxide Dismutase in Regulating Fasting Blood Glucose Levels in Mice Fed a Low versus High Fat Diet
Authors: Natarajan Gopalakrishnan , Perriotte-Olson Curtis , Manickam Devika , Kabanov Alexander , Desouza Cyrus , Viswanathan Saraswathi
Published: June 2021

We previously reported that nanoformulated copper/zinc superoxide dismutase (nanoSOD) ameliorates adipose tissue inflammation without altering systemic glucose homeostasis in a mouse model of diet-induced obesity. A recent report showed that mice lacking SOD1 exhibit insulin resistance only upon low fat (chow) but not high fat (HF)-diet feeding suggesting that the effect of nanoSOD in modulating systemic glucose levels may depend on the diet type. The objective of this study is to determine the effectiveness of nanoSOD in altering muscle gene expression and/or systemic glucose handling in mice fed a low versus high fat (HF) diet. Six-eight wk old wild type C57BL/6 mice were fed a low fat chow diet (CD) or a HF fat (45%) for 10 wk. The mice were injected with nanoSOD (1000 units/kg body wt.) once in two days for fifteen days. The fasting blood glucose level was significantly reduced in CD+NanoSOD-treated mice compared to CD control (
P
<0.05)
.
Insulin tolerance test revealed that nanoSOD-treated mice showed improved glucose handling in response to insulin in CD but not in HF diet-fed mice. The muscle mRNA expression of
LPL
, a gene involved in fatty acid uptake, was significantly increased (
P
<0.05) and
ACOX-1
, a fatty acid oxidation gene, showed a trend towards an increase (
P
<0.052) upon nanoSOD treatment in HF-diet fed mice. On the other hand, genes involved in fatty acid metabolism including
ACOX1, CPT1a
, and
CPT2
were significantly down-regulated in CD+nanoSOD treated mice. Moreover, the expression of
FASN
(
P
<0.05) and
SREBP1
(
P
<0.01), genes promoting fatty acid synthesis was significantly reduced in CD+nanoSOD treated mice. Further, the mRNA expression of
PCX
which promotes both gluconeogenesis and lipogenesis was significantly reduced (
P
<0.01) in CD+nanoSOD treated mice. Taken together, our data show that nanoSOD exerts differential effects on the muscle expression of genes involved in fatty acid metabolism in low fat versus HF diet-fed conditions. Because fatty acid oversupply is a key mediator of muscle insulin resistance, a switch in nutrient supply favoring fatty acid oxidation during HF diet-feeding may have a role in mediating the differential influence of nanoSOD in modulating systemic glucose homeostasis in low versus HF diet-fed conditions.
Abstract 214: Arterial Stiffness in Patients with Metabolic Syndrome: a Cross-Sectional Study in Guayaquil, Ecuador
Authors: Peñaherrera Carlos A , Peñaherrera Ruben E , Duarte Maria C , Peñaherrera Ernesto
Published: June 2021

Background:
metabolic syndrome (MetS) can chronically affect blood vessels, leading to cardiovascular disease. Techniques that assess arterial rigidity and age such as Arteriograph® can provide insight on blood vessel function. We aimed to evaluate arterial stiffness in patients with MetS.

Methodology:
cross-sectional study at Luis Vernaza hospital between November and December 2015. We included patients from the Cardiology Department with a full blood panel. We obtained clinical data for age, gender, history of smoking, hypertension, diabetes, and left ventricular hypertrophy, and measured abdominal girth and blood pressure. Using the Arteriograph®, we measured brachial and central augmentation indexes, central blood pressure, pulse wave velocity (PWV) and arterial age. We retrieved values for triglycerides, HDL, and glycemia from laboratory data. We used IDF criteria to diagnose MetS, but ALAD criteria were used to detect increased abdominal girth. A p value of <0.05 accepted for significance.

Results:
95 patients were selected for analysis. 42 (44.2%) were female and 53 (55.8%) were male, with mean age of 61.7 years (SD 13.1). 58 (61.1%) of our patients had hypertension, 29 (30.5%) had diabetes, and 33 (34.7%) had ventricular hypertrophy. 47 (49.5%) of our patients had MetS. We found significant differences in arterial measurements in MetS vs. non-MetS patients, with higher values in the former (Table 1). We obtained non-significant results for the relationship between arterial age and MetS (p=0.32), even after adjusting for smoking (p=0.28) or ventricular hypertrophy (p=0.71). We found non-significant correlation between age and PWV (p=0.32), and found MetS to be significantly more common in women (p<0.001).

Conclusion:
MetS patients have a higher grade of arterial stiffness. Arterial age was not related to MetS, and it was more prevalent in women. Arteriograph® measurements are helpful to improve diagnosis and management of cardiovascular patients.
Abstract 219: Vascular PTH1R Signaling Limits Aortic Collagen Deposition in Diabetic LDLR-/- Mice
Authors: Cheng Su-Li , Behrmann Abraham , Mead Megan , Ramachandran Bindu , Kapoor Kapil , Perera Ranjan , Kronenberg Henry , 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 arteriosclerosis. The PTH receptor (PTH1R) is highly expressed in the vasculature. To better understand the role of PTH1R signaling in aortic disease, we generated SM22-Cre;PTH1R(fl/fl) mice (PTH1R-VKO) on the LDLR-/- background. No differences in diet-induced diabetes were observed between PTH1R-VKO and Cre-negative PTH1R(fl/fl);LDLR-/- mice (CON). As compared to CON, PTH1R-VKO mice exhibited increased aortic collagen (25.0+/-4.0 vs 15.4+/-1.0 ug collagen / mg aortic dry weight; p = 0.02; n = 9 mice/genotype) but without increased calcification. Aortic pulse wave velocity was not increased but actually decreased in PTH1R-VKO mice (3.7+/-0.3 vs. 4.6+/-0.3 m/sec, p = 0.04, n = 6/genotype). Aortic adventitial myofibroblast cultures from PTH1R-VKO elaborated more collagen in vitro (53.6 +/- 4.9 ug collagen / 250,000 cells vs. 21.7 +/- 5.0 ug collagen / 250,000 cells, p = 0.01). RT-qPCR analysis identified up regulation Col1A1, Col1A2, & Col3A1. To begin to explore the mechanisms whereby PTH1R signaling controls aortic collagen gene expression, we performed expression arrays to profile mRNAs and long noncoding RNAs (lncRNAs) in diabetic PTH1R-VKO and CON cohorts (n = 5/ genotype). A large number of genes encoding myocyte contractile functions (smoothelin-like proteins, alpha-actinins, muscle creatine kinase, ryanodine receptors) were down regulated, while genes encoding collagens and 4- prolyl hydroxylase were significantly increased. A prototypic lncRNA, H19 — initially characterized in stem cells, fetal vascular smooth muscle and neointima — was down regulated in aortas of diabetic PTH1R-VKO mice (90%; p < 0.001) Conversely, the constitutively active PTH1R transgene upregulated H19 5-fold (p < 0.05) in aortic adventitial myofibroblasts. Knockdown of H19 phenocopied actions of PTH1R-deficiency, characterized by the up regulation of Col1A1, Col1A2, and Col3A1. Thus, PTH1R signaling restricts the expression and synthesis of collagen in aortic adventitial cels, mediated in part via H19. Vascular perturbations in PTH1R signaling tone and H19 expression in the aortic adventitia may contribute to the vascular disease of T2D.
Abstract 228: A Novel Combinatorial Non-viral Vector to Treat Familial Hypercholesterolaemia
Authors: Kerr Alastair G , Tam Lawrence , Cioroch Milena , Hale Ashley , Douglas Gillian , Channon Keith , Wade-Martins Richard
Published: June 2021

Familial hypercholesterolaemia (FH) is a life-threatening genetic disorder characterised by elevated levels of plasma low density lipoprotein cholesterol (LDL-C). Loss-of-function mutations in the gene encoding the low density lipoprotein receptor (LDLR) are responsible for ~85% of all FH cases. Current attempts at gene therapy for FH have been limited by the use of strong heterologous promoters which lack regulatory elements essential for controlled expression. We have previously generated mini-gene vectors carrying the human
LDLR
cDNA, driven by 10 kb of genomic DNA from the native human
LDLR
locus, encompassing a promoter region with all essential elements required for physiologically regulated expression (pLDLR-LDLR). We demonstrated that incorporation of the genomic DNA promoter elements resulted in long-term physiologically-regulated
LDLR
transgene expression that complemented Ldlr deficiency. Here we further enhance
LDLR
transgene expression by characterising and cloning in a miRNA, targeting
Hmgcr
(miR82) generating a combinatorial RNAi-LDLR vector (pLDLR-LDLR-miR82). We show in vivo that the combinatorial vector efficiently suppresses endogenous Hmgcr transcripts, which leads to an increase in
LDLR
transgene expression through induction of the
LDLR
promoter. In a preliminary study the pLDLR-LDLR-miR82 vector was able to significantly reduce total and LDL-C, in
Ldlr
-/-
mice fed a 1% cholesterol diet at two and four weeks post vector delivery. We then carried out a longer term study in
Ldlr
-/-
mice fed a 0.25% cholesterol diet, LDLR expression could be detected 12-weeks post-delivery with the plasmid able to be rescued as a functioning episome. LDL-C was significantly lowered throughout the study and this resulted in reduced atherosclerosis in the pLDLR-LDLR-miR82 vector treated mice. Here we demonstrate for the first time, that an episomal non-viral vector is able to significantly reduce LDL-C and the progression of atherosclerosis in a mouse model of FH.
Abstract 232: High-amount and High-intensity Exercise Training Improves HDL Cholesterol Efflux Capacity
Authors: Sarzynski Mark A , Slentz Cris A , Apolzan John W , McGarrah Robert W , Harris Melissa , Church Timothy S , Martin Corby K , Kraus William E , Rohatgi Anand
Published: June 2021

Objective:
Cholesterol efflux capacity (CEC) is associated with incident and prevalent CVD. The effects of regular exercise on CEC are less well known. Thus, we examined the effects of different doses of exercise on CEC in two randomized clinical exercise trials.

Methods:
CEC was assessed before and after six months of exercise training in two cohorts: STRRIDE-PD (N=106) and E-MECHANIC (N=90). In STRRIDE-PD, sedentary, overweight/obese adults with pre-diabetes (fasting plasma glucose 95-125 mg/dL) were randomized to one of four exercise groups: 1) Low-amount/Moderate-intensity (10 kcal/kg/week (KKW) at 50% VO
2
peak); 2) Low/Mod/Diet: Exercise same as group 1 + goal of 7% weight loss; 3) High-amount/Moderate-intensity (16 KKW at 50% VO
2
peak), and 4) High-amount/Vigorous-intensity (16 KKW at 75% VO
2
peak). E-MECHANIC randomized sedentary, overweight/obese, non-diabetic (fasting plasma glucose 76-106 mg/dL) adults into one of two exercise groups (8 KKW or 20 KKW, both at 65-85% VO
2
peak) or a control group. CEC was assessed by measuring efflux of
3
H-labeled cholesterol from J774 macrophages to apoB-depleted plasma (STRRIDE-PD) or serum (E-MECHANIC) over 4 hours. In STRRIDE-PD, only global CEC (+cAMP) was measured; global, non-ABCA1 (-cAMP), and ABCA1 CEC were assessed in E-MECHANIC.

Results:
In STRRIDE-PD, global CEC significantly increased in the High-amount/Vigorous group compared to the other groups, in which CEC actually decreased (
Figure 1A
). In E-MECHANIC, only non-ABCA1-CEC significantly increased in the 20 KKW group compared to controls, with no change in the 8 KKW group (
Figure 1B
). Exercise did not affect global CEC or ABCA1-CEC in E-MECHANIC.

Conclusions:
Within two randomized trials, regular endurance exercise improved CEC only when a high amount of vigorous exercise was performed. Thus, regular vigorous exercise may represent a therapeutic approach for improving HDL function.
Abstract 238: ABCA1 and HDL3 are Required to Modulate Smooth Muscle Cells Trandifferentiation in a Myocardin-miR143/145-dependent Process
Authors: Castiglioni Silvia , Vettore Alessio , Arnaboldi Lorenzo , Calabresi Laura , Corsini Alberto , Bellosta Stefano
Published: June 2021

Cells of the artery wall may accumulate free cholesterol and cholesteryl esters becoming foam cells. Up to 50% of foam cells in human lesions originates from smooth muscle cells (SMCs). Arterial SMCs express the ATP binding cassette (ABC) transporter ABCA1 and, upon cholesterol loading, express macrophage markers and a phagocytic activity. To characterize the role of ABCA1 and HDL3 in this transdifferentiation process, we evaluated the phenotypic changes in SMCs isolated from wild type (WT) and ABCA1 knock out (KO) mice and how HDL3 affects these changes. Cholesterol loading causes the downregulation of the expression of SMC markers including ACTA2, alpha-tropomyosin and myosin heavy chain and increases the expression of macrophage-related genes such as CD68, Mac-2, SRB1, MMPs, ABCG1 and ABCA1. HDL3 treatment in WT cells is able to normalize the expression of ACTA2, while the expression of macrophage-related genes is reduced. On the contrary, the preventive effect of HDL3 is completely lost in ABCA1 KO cells. Interestingly, the presence of HDL3 does not differently affect neutral lipid accumulation in WT or ABCA1 KO cells but stimulates phospholipids removal only in WT cells. ApoAI addition does not reverse the phenotypic changes induced by cholesterol not only in KO but also in WT cells. Moreover, cholesterol loading reduces the expression of myocardin, the master SMC specific-transcriptional coactivator involved in SMC differentiation, by up to 55% (p<0.01 vs respective control) in both cell types. HDL3 normalizes myocardin levels in WT cells while it does not have any effect in ABCA1 KO cells. Similar results are obtained evaluating the levels of miR-143/145, which positively regulate myocardin. The basal expression level of KLF4, a myocardin repressor, is almost double in ABCA1 KO cells compared to WT. After cholesterol loading, KLF4 is slightly reduced in WT cells, while its expression is halved in ABCA1 KO cells. HDL3 restores KLF4 to basal levels in KO cells, but it further reduces them in WT cells. These results indicate that HDL3, modulating the miR143/145-myocardin axis in SMC, prevents the cholesterol-induced gene expression modification regardless of its cholesterol unloading capacity and the presence of ABCA1 is required.
Abstract 24: NETosis is Associated with Abdominal Aortic Aneurysm Rupture
Authors: English Sean J , Albadawi Hassan , Yoo Hyung-Jin , Martinod Kimberly , Diaz Jose A , Meher Akshaya , Upchurch Gilbert R , Wagner Denisa , Watkins Michael T
Published: June 2021

Objectives:
Abdominal aortic aneurysm (AAA) rupture (RAAA) is associated with high mortality. Intraluminal thrombus and associated inflammation have been implicated in RAAA. We sought to characterize neutrophil extracellular trap (NET) formation, or NETosis, associated with a rat RAAA model that we previously developed.

Methods:
To stimulate AAA formation, infrarenal abdominal aortas of male SD rats were infused with porcine pancreatic elastase (PPE): active (AAA, n=21) and heat-inactivated (control, n=14). Aortas were harvested 3, 6, and 14d post PPE exposure. To stimulate RAAA, a separate group of animals was administered β-aminopropionitrile (BAPN, n=12) daily. Those animals that did not rupture (NRAAA-14d, n=6) were harvested 14d post PPE exposure. Paraffin embedded sections were analyzed using immunofluorescence. To evaluate evidence of primarily neutrophil activity and NET generation, myeloperoxidase (MPO) and peptidylarginine deiminase 4 (PAD4) were quantified utilizing Cy3 and Alexa-488 secondary antibodies, respectively. To determine the presence of NETs, citrullinated histone H3 (H3Cit, Cy3) was visualized. DAPI was utilized to detect nuclei. Aortic sections were imaged with fluorescent microscopy, and quantification of percent area stained (PAS) was performed by threshold analysis.

Results:
Greater MPO PAS was observed for AAA-6d compared with control-6d (p=0.03). Greater PAD4 PAS was observed for AAA-3d (p=0.02) and -6d (p=0.05) compared with control-3d and -6d, respectively. Greater H3Cit PAS was observed for AAA-6d (p<0.0001) and -14d (p=0.001) compared with control-6d and -14d, respectively. In the setting of rupture stimulation, greater H3Cit PAS was observed for RAAA compared with AAA-14d (p=0.001) and NRAAA-14d (p=0.0002).

Conclusions:
Increased PAD4 expression by AAAs compared with controls at 3 and 6d post PPE exposure, results in increased H3Cit expression at 6 and 14d post PPE exposure. Comparable H3Cit expression by AAA-6d and RAAA demonstrates the acute nature of this rupture model; however, greater H3Cit expression by RAAAs suggests a role for NETosis in RAAA. Further evaluation of NETosis as it pertains to human RAAA is necessary. Therapeutic inhibition of NETosis may slow AAA progression and prevent RAAA.
Abstract 254: Platelet Derived Growth Factor Receptor Beta Activation Promotes Atheroprotective Changes in Smooth Muscle Cell Phenotype
Authors: Newman Alexandra A , Cherepanova Olga A , Owens Gary K
Published: June 2021

Despite decades of research, little is known about mechanisms and factors driving atherosclerotic progression leading to plaque rupture, thrombosis and myocardial infarction or stroke. The widely accepted dogma is a thin fibrous cap and a paucity of Acta2+ smooth muscle cells (SMC) relative to macrophages increases risk of plaque rupture. However, our recent rigorous lineage tracing studies in advanced lesions showed a subset of cells that express macrophage markers are SMC-derived and SMC specific conditional knockout of stem pluripotency gene Klf4 resulted in increased fibrous cap thickness and decreased lesion size and abundance of SMC-derived macrophage-like cells. Results indicate that SMC play a critical role in lesion pathogenesis but paradoxically can transition to a beneficial or detrimental state, presumably as a function of the local environmental milieu within lesions. Studies highlight the critical need to identify factors and mechanisms that direct SMC to form a stable fibrous cap. Various
in vitro
data show that SMC treated with platelet derived growth factor receptor beta (PDGFR beta) ligands dedifferentiate, proliferate, and synthesize extracellular matrix (ECM) molecules, including collagens known to be enriched within the fibrous cap. We hypothesize that PDGFR beta activation in SMC is necessary for fibrous cap stabilization through EMC protein synthesis. Herein we employ Myh11-ER
T2
-cre eYFP SMC lineage tracing ApoE-/- Western diet fed mice to show that a subset of SMC in advanced lesions express PDGFR beta and are localized to the fibrous cap. Moreover we demonstrate that SMC specific conditional KO of the PDGFR beta in these lineage tracing mice resulted in dramatic reductions in the numbers of YFP+ (SMC-derived) cells within lesions but an increase in YFP- (not SMC-derived) macrophages, as well as a decrease in Acta2+ cells in the fibrous cap. Results indicate that PDGFR beta activation in SMC is a critical determinant of the cellular composition of lesions and is essential for investment of SMC within the fibrous cap. Supported by NIH R01 grants HL121008 and HL087867 to GKO.
Abstract 255: Plasminogen Promotes Cholesterol Efflux by the ABCA1
Authors: Pamir Nathalie , Dichek David A , Getz Godfrey S , Marcovina Santica , Heinecke Jay W
Published: June 2021

Background:
The cholesterol efflux capacity (CEC) of serum HDL, measured using cultured macrophages predicts incident and prevalent CVD risk in humans. The ABCA1 pathway is a key regulator of macrophage cholesterol homeostasis in vivo.

Methods:
We used genetic and biochemical approaches in mice to identify important mediators of CEC.

Results:
On high-resolution size-exclusion chromatography of mouse plasma, macrophage CEC and HDL co-eluted as a single major peak, suggesting that HDL mediates cholesterol efflux. In contrast, size-exclusion chromatography revealed two major peaks of material that promoted ABCA1-specific CEC, one of which was distinct from HDL. HDL particle concentration was reduced by 75% in

Apoa1
-/-

mice; this resulted in a 50% decrease in macrophage CEC but, surprisingly, had no impact on ABCA1-specific CEC. Orthogonal chromatography-mass spectrometric analysis of the non-HDL-associated efflux inducing material isolated from wild-type and APOA1 deficient plasma showed that plasminogen strongly correlated with ABCA1-specific CEC. Moreover, isolated plasminogen promoted cholesterol efflux by the ABCA1 pathway, and the specific activity of ABCA1-specific CEC of non-HDL-associated material was reduced by 50% in plasminogen deficient plasma. Imaging of cells treated with fluorescently-labeled antibodies demonstrated that ABCA1 and plasminogen co-localized on the plasma membrane.

Conclusions:
HDL particle concentration is an important contributor to macrophage CEC. However, other pathways contribute to ABCA1-specific CEC; our studies identify plasminogen as one potential mediator. Plasminogen associates with CVD risk in human genetic studies, raising the possibility that it plays a role in atherosclerosis by modulating ABCA1-mediated sterol efflux from macrophages.
Abstract 264: Oral Activated Charcoal Adsorbent (AST-120) Inhibition of Chronic Kidney Disease-induced Atherosclerosis Involves Modulation of Intestinal Immunity
Authors: Tsuchida Yohei , Wilheln Ashley , Major Amy , Davies Sean , Linton Macrae , Kon Valentina
Published: June 2021

Introduction and Aims:
Although patients with chronic kidney disease (CKD) are at the highest risk for atherosclerotic cardiovascular disease (CVD), current interventions (e.g. statins) that are consistently effective in other high-risk groups have been found to be insufficiently effective in CKD. Serum levels of uremic toxins including indoxyl sulphate have been implicated in cardiovascular mortality. The oral charcoal absorbent AST-120 reduces serum levels of indoxyl sulphate through adsorption of indole converted from dietary tryptophan in the gastrointestinal tract. Previously, we reported the inhibitory effects of AST-120 on progression of atherosclerosis in apoE-deficient hyperlipidemic mice. Because gut immunity can affect systemic inflammatory responses and atherogenesis, we hypothesized that the AST-120-mediated benefits in CKD-acceleration of atherosclerosis involve modulation of intestinal immune cells.

Methods:
Eleven week old apoE-deficient mice underwent a reduction in renal mass through subtotal nephrectomy (sNx, n=34) or sham-operation (S, n=14). Two weeks later, each of the 2 groups was further divided into AST-120-treated or untreated mice. Six weeks later, we used flow cytometry and real time PCR to examine the population of immune cells in intestinal Peyer’s patches and mesenteric lymph nodes.

Results:
AST-120 had no significant effect on cellular populations of Peyer’s patch or mesenteric lymph nodes in mice with intact kidneys. Interestingly, however, in sNX mice treatment with AST-120 significantly decreased numbers of Peyer’s patch cytotoxic T cells (-19.6%; P=0.008), CD8
+
central memory T cells (-33.9%; P=0.014), and CD8
+
naïve T cells (-42.6%; P=0.004) compared to controls. In mesenteric lymph nodes of sNX animals, AST-120 did not affect CD4
+
T helper cell number, but reduced early activated CD4
+
T cells (-13.1%; P=0.04). Cytokine expression in mesenteric lymph nodes of AST-treated sNX mice showed increases in TGF-β (+47.2%; P=0.022) and IL-10 (+36.5%).

Conclusions:
The mechanism underlying the ability of oral activated charcoal adsorbent AST-120 to inhibit the acceleration of atherosclerosis in the setting of CKD may involve modulation of immune cells in the gut mucosa.
Abstract 268: Vascular Dysfunction in High BMI Black College Football Players
Authors: Powers Edward M , Fudim Murat , Silver Heidi J , Fitch Robert W , Niswender Kevin D
Published: June 2021

Background:
Former football players (FP) who competed with BMI > 30 have high rates of atherosclerosis and early mortality (28% by age 50). FP are disproportionately black, a group predisposed to hypertension and atherosclerosis.

Hypothesis:
Positive energy balance and oxidative stress lead to vascular dysfunction in black FP.

Methods:
High BMI college FP (n=33) underwent metabolic and vascular testing at the Vanderbilt Medical Center during the offseason training program. Endothelial function was tested using flow-mediated dilation (FMD) of the brachial artery. Arterial elasticity and vascular resistance were tested using a calibrated tonometer. Regression was performed using least squares on Stat, version 12.

Results:
Elevated blood pressure (EBP) (SBP > 130) was common in both black (n = 14) and white (n = 19) athletes (78% vs 63%, p = .34). Black players had significantly higher systemic vascular resistance and lower arterial elasticity. However, they had significantly better lipid profiles and body composition, and comparable insulin resistance assessed by HOMA.

In black FP, EBP was associated with positive energy balance (4.3 kg gained during six weeks from enrollment to clinic visit vs. 0.7 kg, p = .05). Daily caloric intake predicted endothelial function as measured by flow mediated vasodilation (FMD) (r=.76, p=.001). Caloric intake and oxidative stress (F2-isoprostanes) trended to inversely correlate with larger artery elasticity (r=.40, p=.09 and r=.41, p=.09, respectively). HOMA did not predict FMD (r < .01, p = .56).

Respiratory quotient (RQ) correlated with f2-isoprostanes (r=.53, p=.02), suggesting a link between mitochondrial dysfunction and oxidative stress.

Conclusion:
High BMI black FP suffer from vascular dysfunction, possibly due to oxidative stress from overfeeding. This correlates with studies of non-athlete adult population, but differs notably in being independent of insulin resistance. A larger, longitudinal study is needed to establish a link between overfeeding, vascular dysfunction and early cardiovascular morbidity and mortality in high BMI black athletes. The role of oxidative stress and selective use of nitric oxide donor drugs in black athletes should be explored.
Abstract 272: Depressed Individuals May Not be Aware of Their Own Dyslipidemia
Authors: Ramirez Francisco E , Siebold Jill , Antuna Katelyn , Sanchez Albert , Nedley Neil
Published: June 2021

Background:
Awareness of dyslipidemia can help persons motivate and maintain better blood lipid control.

Hypothesis:
Self perceived cholesterol levels may not match the real lab proven results.

Methods:
To date, 368 patients completed a residential depression program. Every patient answered several mental tests, including a depression and anxiety recovery program assessment test, at baseline and at the end of the program. One question on the test is, "Do you have high cholesterol?” Patients can answer: No, Yes, or I don't know. Each patientundergoes complete blood work, including lipids. Blood analysis is done by LabCorp, Inc. We used LabCorp range for total cholesterol which is 100 to 199 mg/dl. Finally, laboratory results were compared to patients’ questionnaire answers to evaluate the accuracy of their pre-lab estimation.

Results:
Of the 368 patients, 238 (64%) were females, 80% were Caucasian, 8% were Hispanic, 3.5% African American and 2% were Asian. According to laboratory results, 274 (74%) patients had normal cholesterol and 94 (26%) had high cholesterol. The high cholesterol group had an average cholesterol level of 234 mg/dl (SD 32.4, SEM 3.3). The normal cholesterol group had an average cholesterol level of 154 mg/dl (SD 26, SEM 1.5). Of those with high cholesterol, 39% (n=37) thought they did not had high cholesterol, 14.8% (n=14) answered that they did not know if they had high cholesterol and 47.7% (n=43) answered “Yes”, recognizing that they had high cholesterol. For those with normal cholesterol levels, 81.3% (n=223) correctly estimated that they did not have high cholesterol, 10.9% (n=30) did not know if they had high cholesterol, and 7.6% (n=21) believed they had high cholesterol when in reality their levels were normal.

Conclusion:
Almost half of the depressed patients (48%) were not aware of their high total cholesterol. It would be interesting to see if this pattern is also true also for those that are not depressed. Health providers should take notice that many depressed patients are unaware of their high cholesterol levels.
Abstract 3: Targeting Macrophage Necroptosis for Therapeutic and Diagnostic Interventions to Treat Atherosclerosis
Authors: Karunakaran Denuja , Geoffrion Michele , Wei Lihui , Gan Wei , Perisic Ljubica , Maegdefessel Lars , Hedin Ulf , Sad Subash , Ruddy Terrence , Guo Liang , Kolodgie Frank , Virmani Renu , Rayner Katey J
Published: June 2021

Background:
Atherosclerosis results from maladaptive inflammation driven primarily by macrophages, whose recruitment and proliferation drive plaque progression. In advanced plaques, macrophage death contributes centrally to the formation of plaque necrosis, which underlies the instability that promotes plaque rupture and myocardial infarction. As such, targeting macrophage cell death pathways may offer promise for the stabilization of vulnerable plaques. Necroptosis is a recently discovered pathway of programmed cell necrosis regulated by RIP3 and MLKL kinases that in contrast to apoptosis, induces a pro-inflammatory state. We hypothesize that atherogenic ligands within the plaque promote macrophage necroptosis and this process underlies necrotic core formation and drives atherosclerotic plaque instability.

Results:
In humans with unstable carotid atherosclerosis, expression of RIP3 and MLKL is increased and MLKL phosphorylation, a key step in the commitment to necroptosis, is detected in advanced atheromas. Investigation of the molecular mechanisms underlying plaque necroptosis showed that macrophages treated with oxidized LDL have increased expression of necroptotic genes RIP3 and MLKL through ROS-dependent activation of the promoter region and increased RIP3 and MLKL phosphorylation. Combined treatment with oxLDL and DAMPs (damage associated molecular patterns) amplified macrophage necroptotic cell death, indicating that additional inflammatory stimuli present in the lesion could act synergistically to promote necroptosis. Using a radiotracer developed with the necroptosis inhibitor Nec-1, we show that
123
I-Nec1 localizes specifically to atherosclerotic plaques in
Apoe-/-
mice, and its uptake is tightly correlated to lesion areas by
ex vivo
nuclear imaging. Furthermore, treatment of
Apoe-/-
mice with established atherosclerosis with Nec-1 reduced lesion size and markers of plaque instability, including necrotic core formation.

Conclusions:
Our findings offer molecular insight into the mechanisms of macrophage cell death that drive necrotic core formation in atherosclerosis and suggest that this pathway can be used as both a diagnostic and therapeutic tool for the treatment of unstable atherosclerosis.
Abstract 302: Prevalence of Acquired Long QT Syndrome in Patients with Aortic Valve Stenosis
Authors: Khaji Amanulla , Waqas Khan Hafiz M , Shaik Zakir , Fityani Ahmed , Gnall Eric , Rodriguez Roberto , Abramson Sandra , Evenson Deborah L , Plestis Konstadinos , Goldman Scott , Kowey Peter R , Zhang Li
Published: June 2021

Introduction:
QT prolongation is associated with an increased risk of sudden cardiac death (SCD). Patients (pts) with aortic valve stenosis (AVS), in particular, have a higher incidence of SCD. We hypothesized that cardiac remodeling in the structural heart abnormalities may result in secondary QT prolongation in AVS.

Methods:
Electronic medical records in AVS pts prior to valve repairs/replacement in a single center from 2005-2015 were retrieved. ECGs showing QRS duration (QRSD) > 120 ms or non-sinus rhythm were excluded for QT evaluations. Information regarding concomitant hypertension (HTN), dyslipidemia, diabetes, coronary artery disease, heart failure and presence of QT-prolonging factors (electrolytes imbalance and using QT prolonging drugs) was also evaluated.

Results:
Among 419 AVS pts [median age 75 (13) yrs, male 56%] with normal QRSD [94 (16) ms], the prevalence of acquired long QT syndrome with and without known QT-prolonging factors is shown in Table 1. More pts with heart failure had QTc ≥ 450 ms than those without HF (64% vs. 48%, p <0.01).

Conclusions:
Regardless of the known QT prolonging factors, the prevalence of QT prolongation is high in AVS and may serve as a pathogenetic factor for sudden death in patients with aortic valve stenosis.
Abstract 308: Aortic Valve Stenosis Causes Both Delayed Ventricular Depolarization and Repolarization
Authors: Shaik Zakir , Khaji Amanulla , Goldman Scott , Rodriguez Roberto , Sutter Francis P , Waqas Khan Hafiz M. , Alfityani Ahmed , Gnall Eric , Coady Paul , Plestis Konstadinos , Kowey Peter R , Zhang Li
Published: June 2021

Background:
Increased QRS duration (QRSD), a common ECG finding in patients (pts) with aortic valve stenosis (AVS), can result in false QT prolongation. This study aimed to test a hypothesis that in AVS delayed ventricular repolarization is independent from the delayed depolarization.

Methods:
In a retrospective AVS study, ECGs prior to valve repair/replacements were evaluated. QRSD and QTc (Bazett's) were compared between pts with severe and non severe AVS. ECGs showing QRSD > 120 ms or non-sinus rhythm were excluded for analysis. In pts with both wider QRS and longer QT interval (QTc > 440 ms), JT (QT-QRSD) was used to determine the repolarization time.

Results:
Pts with severe AVS (n=219) had longer QRSD [100 (16) ms vs. 88 (12) ms, p<0.001], with QTc ≥ 450 ms seen in 53%. JT is much longer in the ALQTS group (Table 1). More pts had QTc ≥ 470 ms (33% vs 23%, p<0.05) in the severe AVS than the non-severe AVS.

Conclusion:
A wider QRS and higher prevalence of moderate to markedly prolonged QT interval in pts with severe AVS indicates AVS itself can result in a delay of both ventricular depolarization and repolarization.
Abstract 309: A Female

Myh11-CreER
T2

Mouse Line Resulting from Y to X Chromosome-translation
Authors: Zhou Junmei , Liao Mingmei , Yang Pu , Ali Yasmin , Zou Fei , Wang Fen , Jiang Zhihua
Published: June 2021

The

Myh11-CreER
T2

mouse line (

Cre
+/

) provides a powerful tool for studying the role of smooth muscle cells (SMCs) in the pathogenesis of vascular diseases. The
Cre
allele was initially inserted into Y chromosome, and thus excluded its inheritance by female mice. However, a number of vascular diseases exhibit sexual dimorphism in development and progression, and the underlying mechanisms may involve regulation of SMC biology by the dosage-effects of sex-linked genes and gender. A
Cre
line that induces SMC-specific gene deletion in female mice would complement the existing

Cre
+/

line to address this critical issue. Our lab established the

Cre
+/

colony with one male ancestor and it spontaneously separated to two subcolonies during subsequent breeding. One subcolony produced only male

Cre
+/

progenies, but the other produced both male and female

Cre
+/

progenies, indicating chromosome-translocation of the Cre allele. In view of this observation, we mapped the chromosome-location of the
Cre
allele for these subcolonies with cross-breeding experiments. The 1
st
experiment crossed two

Cre
+/

males from the first subcolony with four

Cre
-/

females. Of the produced progenies, all males (
n
=9) carried and all females (
n
=22) did not carry the
Cre
allele, which favors Y chromosome- (

Y
Cre+

) over autosome-location of the
Cre
allele in this subcolony (Bayes Factor>1000). In the 2
nd
experiment, a

Cre
+/

female from the second subcolony was bred with a

Cre
-/

male. Then, a

Cre
+/

male progeny of these breeders was mated with a

Cre
-/

female. The former breeding pair exhibited equal inheritance of the
Cre
allele by its male and female offspring. The latter breeding pair showed that all female progenies (
n
=8) did, but all male progenies (
n
=4) did not, carry the
Cre
allele. The data favors X chromosome- (

X
Cre+

) over autosome-location of the
Cre
allele in this subcolony (Bayes Factor>1000). To assess the recombination driven by

Cre
+/

allele with different chromosome-location, we crossed

X
Cre+

and

Y
Cre+

strains with
R26R
reporter mice. Robust Cre activity (x-gal staining) was observed in vascular SMCs of

Y
Cre+

and

X
Cre+

mice. The

X
Cre+

mice generated via Y to X chromosome-translocation thus represents a novel strain that is suitable for inducible gene deletion in SMCs of female mice.
Abstract 314: Development of a Nanotube-coated Nitinol Stent for Delivery of Resolvin D1
Authors: Lee Phin-Peng , Chatterjee Anuran , Wu Bian , Nuhn Harald , Desai Tejal , Conte Michael S
Published: June 2021

Introduction:
As part of a Vascular Innovation and Therapeutic Advances (VITA) contract from NHLBI, we sought to optimize a nitinol drug eluting stent to deliver a novel bioactive lipid mediator Resolvin D1 (RvD1).

Methods:
Nitinol stents were secured as the anode in an ethylene glycol-based anodization setup to create nanotubes on the stent surface. Scanning electron microscopy was used to evaluate the quality of the coatings where an iterative process was utilized to optimize anodization conditions (70V, 5min). To ascertain structural integrity of the coatings, stents were observed under SEM after compression in a crimping device followed by self-expansion. Nanotube-coated (NT) and unmodified bare metal (BM) nitinol stents were loaded with RvD1 by dip-coating (with sonication) followed by hanging drop evaporation of RvD1 (in ethanol). Stents were eluted in media and RvD1 levels were analyzed by EIA. An
ex vivo
model of vascular stent implantation was created using a 3D printer and RvD1 delivery to the vessel wall (segment of rabbit aorta) and circulating flow medium was quantified after 3 hrs of stent implantation.

Results:
NT stents released significant amounts of RvD1 (1273ng cumulative at day 9), where 1177ng (± 238) of RvD1 was released within 3hr and 0.111ng (± 0.014) of RvD1 was released between day 7 and day 9. Net amount of RvD1 released was 0.2nM - 2000nM, which is physiologically relevant and within the milestone goal. NT stents released much higher levels of RvD1 (9.7 pg/mg vessel; ±1.39) to the vessel wall compared to standard BM stent controls (3.9 pg/mg vessel; ±2.0) and native vessel (1.3 pg/mg vessel; ±0.5).

Conclusion:
We have developed optimized anodization conditions for creating a uniform coating of titania nanotubes on nitinol stents that is able to survive crimping and self-expansion. NT stents demonstrate improved loading and elution of the lipid mediator RvD1 in an ex-vivo artery flow model.
Abstract 316: A Novel Mechanism by Which Tumor Necrosis Factor Alpha Protects Mice From Lipopolysaccharide-induced Vasodilatory Shock, Hypotension, and Motility
Authors: Zhao Guogang , Liu Shu , Su Wen , Song Xiulong , Gong Ming C , Guo Zhenheng
Published: June 2021

Objectives:
Sepsis is defined by a severe systemic inflammatory response to infection, and its complications, including vasodilatory shock and hypotension, can be fatal. Vasodilatory shock has long been believed as a result of failure of the vascular smooth muscle cells (VSMCs) to respond to vasopressors to constrict and result in hypotension; however, the mechanism is largely unknown.

Approaches and Results:
Here, we report that protein kinase C (PKC)-potentiated phosphatase inhibitor of 17 kD (CPI-17), a key regulator in smooth muscle contraction, was selectively downregulated by endotoxin lipopolysaccharide (LPS) in mesenteric arteries from C57BL/6 mice and by tumor necrosis factor alpha (TNF) in cultured smooth muscle tissue and cells. Downregulation of CPI-17 by LPS was associated with LPS-induced vasodilatory shock and hypotension and was largely attributed to TNF-induced CPI-17 transcriptional suppression. By various molecular approaches, including cloning, deletion, mutation, siRNA, electrophoretic mobility shift assay (EMSA), and chromatin immunoprecipitation (ChIP), we identified two GC-boxes in the proximal CPI-17 promoter as a novel TNF-response element that is critical for TNF-induced CPI-17 downregulation, and demonstrated that in the absence of TNF, SP1 bound to the CPI-17 promoter GC-boxes and positively regulated CPI-17 transcription, whereas in the presence of TNF, Krüppel-like factor 4 (KLF4) was markedly upregulated, competed with SP1 for binding to the CPI-17 promoter, and negatively regulated CPI-17 transcription through histone deacetylases (HDACs). Moreover, specific blockage of the TNF/KLF4/SP1/CPI-17 signaling by genetic deletion of TNF prevented mice from LPS-induced hypotension and mortality.

Conclusions:
These data reveal a previously unrecognized but potentially important mechanism for vasodilatory shock and hypotension in sepsis, and suggest a new therapeutic strategy for treatment of patients with sepsis.
Abstract 325: Non-Invasive Volumetric Assessment of Angiosome Perfusion in Diabetic Patients With Critical Limb Ischemia and Non-Healing Ulcers Using Radiotracer Imaging
Authors: Buckley Jessica L , Mena Carlos , Papademetris Xenophon , Blume Peter A , Sumpio Bauer E , Sinusas Albert J , Stacy Mitchel R
Published: June 2021

Introduction:
Impaired lower extremity perfusion is a hallmark of peripheral vascular disease (PVD) and is particularly problematic in diabetic patients, who suffer from high rates of PVD, ulceration, and lower extremity amputation. The ability to non-invasively detect deficits in microvascular perfusion within vascular territories, or angiosomes, of the feet may provide information related to tissue viability, assist with classification of diabetic wounds, and guide future therapeutic interventions. In this study, we sought to quantify volumetric microvascular perfusion within specific angiosomes containing non-healing foot ulcers in diabetic patients with critical limb ischemia.

Methods:
Twenty-eight diabetic patients (mean age, 68 ± 13 yrs) with non-healing ulcers underwent SPECT/CT imaging of the feet following a resting injection of
99m
Tc-tetrofosmin (dose, 550.6 ± 37 Mbq). CT images were segmented into five angiosomes and used for quantifying relative radiotracer uptake (Fig. 1A), expressed as standardized uptake values (SUVs). SUVs were assessed for each patient in the angiosome containing the non-healing ulcers, which were rated using the Wagner Grade Classification.

Results:
SPECT/CT imaging allowed for visualization of resting perfusion deficits in ulcer sites (Fig. 1B) and quantitative image analysis revealed variability in angiosome perfusion based on Wagner Grade of ulcers. Angiosomes containing Wagner Grade IV ulcers exhibited significantly lower SUVs when compared to SUVs of angiosomes containing Wagner Grade I, II, and III ulcers (Fig. 1C).

Conclusions:
SPECT/CT imaging provides a useful non-invasive tool for evaluating a wide clinical spectrum of ulcers within associated angiosomes of the foot and can be performed without requiring pharmacological or exercise stress. Future application of SPECT/CT imaging may provide additional value for detection and targeting of ischemic tissue for therapeutic interventions.
Abstract 329: The Role of TIE1 in Flow Mediated Lymphatic Vessel Remodeling and Valvulogenesis
Authors: Harmelink Cristina , Zhou Bin , Qu Xianghu , Baldwin H. Scott
Published: June 2021

Recently, it has been shown that the mechanical stimulus of turbulent shear stress caused by onset of lymph flow is required for lymphatic remodeling, maturation, and lymphatic valve (LV) development. Homeostasis of the adult lymphatic vasculature also relies on flow-mediated signal transduction. However, the cellular machinery responsible for transducing mechanosensory signals required for lymphatic network formation and maintenance is unknown. Our laboratory has previously shown that TIE1 is at least partially responsible for mechanotransduction of turbulent flow required for initiation and maintenance of atherosclerotic plaque formation at the branch points of systemic vasculature in the adult animal. Moreover, TIE1 is expressed throughout lymphatic vasculature during mouse embryogenesis into adulthood, with enrichment in LVs. To circumvent the embryonic lethality caused by global Tie1 disruption, we conditionally deleted Tie1 using Nfatc1Cre. Nfatc1Cre drives recombination in lymphatic endothelial cells, with strong expression in the LVs. Nfatc1Cre:Tie1fl/fl mutants survive to birth but accumulate chyle in the peritoneal and pleural cavities by postnatal day 2. The lymphatic vessels in the mutants are dilated and tortuous, and do not undergo normal hierarchical remodeling. The constrictions that normally indicate intraluminal valve development are lacking in the mutant lymphatic vessels. Underlying these defects in the Nfatc1Cre:Tie1fl/fl mutants is loss of the normal molecular landscape associated with lymphatic patterning and valvulogenesis. Therefore, we hypothesize that Tie1 orchestrates the mechanotransduction necessary for intraluminal LV development and postnatal maintenance.