bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 162: A Novel Nanotechnology Approach for Cholesterol Loading and Reverse Cholesterol Transport Assessment
Authors: Badgeley Marcus A , Rajagopalan Sanjay , Maiseyeu Andrei
Published: March 2022

Objective:
Functional significance and efficiency of reverse cholesterol transport (RCT) pathways of cholesterol efflux is of major pathogenetic and therapeutic relevance. The assays used to test RCT however continue to remain fairly labor and time intensive with the “gold standard” approach in vitro employing acetylated low-density lipoprotein (acLDL)-mediated loading of cells with radioactively labeled cholesterol followed by exposure to a cholesterol acceptor (e.g. high-density lipoprotein, HDL). We tested the feasibility and efficacy of a synthetic CHolesterol Efflux Assay Probe (CHEAP) capable of Förster Resonance Energy Transfer (FRET) as an alternate strategy to quantify RCT that could theoretically replace more labor intensive methods via its advantages of high throughput screening, faster and economical synthesis, bulk fabrication, and real time cholesterol release monitoring.

Methods and results:
CHEAP synthesis was accomplished through self-assembly methodologies from polystyrene core nanoparticles, lipids, and H3- or BODIPY- labeled cholesterol. Screening of 25 potential formulations for multiple physicochemicalproperties including stability, polydispersity, zeta potential and FRET efficiency upon cholesterol release resulted in identification of an optimized particle with mean size similar to LDL (32.3 nm). Fluorescence profiles of BODIPY-cholesterol loaded and cholesterol free CHEAP were characterized to allow real time determination of cholesterol release kinetics ex vivo and in vitro through changes in Förster Resonance Energy Transfer upon cholesterol release. Internalization was studied via confocal microscopy and the results showed that particles localized to early endosomal and lysosomal intracellular compartments. Assay efficacy of CHEAP was investigated in RCT with varying doses of human HDL as cholesterol acceptors. Figure 1 demonstrates that cholesterol loading with CHEAP resulted in dose-dependent cholesterol efflux (r2 = 0.83, p=0.011) as compared with acLDL loading (r2 = 0.67, p=0.048).

Conclusions:
CHEAP has several advantages over lipoprotein cholesterol delivery probes and has reliable results in RCT assays.
Abstract 170: Statin Therapy Increases Plasma Levels of Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) in Hypercholesterolemic Subjects
Authors: Qamar Arman , Ferguson Jane , Khera Amit , DerOhannessian Stephanie , Wolfe Megan , Ky Bonnie , Rader Daniel
Published: March 2022

Background
PCSK9 is a secreted glycoprotein that modulates low density lipoprotein cholesterol (LDL-C) levels by negatively regulating LDL receptor (LDLR). In humans, gain of function mutations in PCSK9 increase LDL-C and loss of function mutations reduce LDL-C levels. We hypothesized that PCSK9 may be regulated by statin therapy.

Objective
The aim of this study was to determine the effect of pravastatin and atorvastatin on plasma PCSK9 levels in healthy subjects with hypercholesterolemia

Methods & Results
Hypercholesterolemic subjects (n=94) were randomized in a double blind, placebo controlled study to pravastatin 40mg/day (n=21), atorvastatin 10mg/day (n=26), atorvastatin 80mg/day (n=25), or placebo (n=22) and treated for 16 weeks. Plasma PCSK9 levels were measured using a sandwich ELISA at baseline and 16weeks. There were no significant changes in plasma PCSK9 in the placebo & atorvastatin 10 mg group. In comparison to baseline, after 16 weeks of treatment we observed a 42 % increase (247 ± 19 ng/ml to 317 ± 19 ng/ml, p value <0.05) with pravastatin 40mg/day, and a 28.4% increase (286 ± 15 ng/ml to 353.5±17 ng/ml, p value <0.05) with atorvastatin 80 mg/day in plasma PCSK9 levels.

Conclusion
In this randomized, placebo-controlled trial, we found a significant increase in plasma PCSK9 levels induced by pravastatin and atorvastatin. These results suggests a possible explanation for diminished LDL-C lowering with increasing doses of statin therapy.
Abstract 187: Differential Coronary Remodeling and Plaque Composition Patterns Manifest in Diabetic Versus Nondiabetic Coronary Artery Disease: Insights from Pre--Percutaneous Coronary Intervention in Vivo Compositional Analysis Using Intravascular Ultrasound Virtual Histology
Authors: Gajjar Mark , Jagadeesan Vikrant , Lee Linda , Nathan Sandeep
Published: March 2022

Background:
Prior reports note patients (pts) with diabetes mellitus (DM) and coronary artery disease (CAD) undergo negative vascular remodeling with vessel shrinkage and low-volume plaque in angiographically stenosed segments. We assessed coronary remodeling and correlated findings with
in vivo
Intravascular Ultrasound Virtual Histology (IVUS-VH) compositional analyses.

Methods:
2,579 IVUS-VH frames acquired in DM (n=15) and non-DM (n=21) pts, pre-PCI via Volcano Eagle-Eye Platinum 20 MHz IVUS and automated pullback were traced, blindly adjudicated and analyzed for remodeling parameters and VH plaque composition. Statistical analyses were performed using SPSS 20.0.

Results:
DM pts had significantly greater coronary remodeling, plaque area and burden but smaller luminal cross-sectional area (CSA) as compared with non-DM pts despite nearly identical reference segment IVUS dimensions and angiographic lesion appearance. (Fig.1). By IVUS-VH, DM plaque contained greater necrotic core (1.87 vs 1.66 mm2/frame, p = 0.002 and fibrous content (4.94 vs 4.4 mm2/frame, p <0.001).

Conclusions:
Contrary to prior reports, we demonstrate DM pts have positive coronary remodeling in response to CAD and to a greater extent than non-DM pts. Observed plaque burden in DM pts was a result of both increased plaque area/frame and decreased luminal CSA. The differential VH plaque composition in DM pts may be linked with the differential remodeling pattern observed. These data have significant implications on pathogenesis and treatment of DM CAD.
Abstract 198: Polymeric Transfection Is Superior to Traditional Transfection Methods in Targeting the Cellular Processes of Intimal Hyperplasia
Authors: Mountain Deidra J , Kirkpatrick Stacy S , Freeman Michael B , Stevens Scott L , Goldman Mitchell H , Grandas Oscar H
Published: March 2022

OBJECTIVE:
We have previously demonstrated that targeted MT1-MMP (MT1) silencing, via pharmaceutical transfection, inhibits the cellular processes of intimal hyperplasia (IH) in vitro. Manipulation of MT1 in vivo should be examined for the prevention of IH, but traditional methods of genetic delivery are poorly translatable in vivo. Biodegradable polymers have shown promise in their ability to safely deliver genetic material in early feasibility studies using stem cells. Our goal is to establish polymeric transfection as an effective method for gene therapy in differentiated vascular cells and as a translatable option for therapeutic gene modulation in the prevention of IH. Here we compared polymeric transfection and traditional pharmaceutical transfection in their efficiency in MT1 silencing and their downstream effectiveness in the inhibition vascular smooth muscle cell (VSMC) migration and proliferation.

METHODS:
VSMCs were transfected with MT1 or negative control (NC) siRNA using DharmaFECT2 complexes (Dharm) or poly(β-amino ester) polymer bioconjugates (StemFECT; Stem).

RESULTS:
Stem was significantly more effective than Dharm in silencing MT1 (% expression vs. NC; Stem=40±3*; Dharm= 58±6%; n=4-7; *P<0.05). Zymography revealed that silencing of MT1 decreased MMP-2 activity similarly in both methods. Silencing of MT1 via Stem decreased migration to 59±6% vs. NC (n=7; P<0.05), while silencing via Dharm only decreased migration to 88±6% vs. NC (n=3). Likewise, silencing of MT1 via Stem inhibited basal and serum-stimulated proliferation to 89±3% and 97±6% vs. NC respectively (FBS=162±6%; n=4-6; P<0.05), while silencing via Dharm did not significantly inhibit basal or serum-stimulated proliferation.

CONCLUSION:
Evidence here demonstrates 1) VSMCs can be genetically altered via polymeric transfection in a manner superior to traditional pharmaceutical transfection and 2) silencing MT1 via polymeric transfection is equally effective in decreasing MMP enzymatic activity and more effective in inhibiting the end cellular responses of IH development in vitro. Polymeric transfection has potential for in vivo delivery of molecular inhibitors of MT1 and others aimed at targeted genetic therapy for vascular disease.
Abstract 206: PPARγ Deficiency Causes Aortic Stiffness and Dysregulated Elastin Turnover
Authors: Tai Haw-Chih , Chen Ju-Yi , Tsai Pei-Jane , Hagaman John R , Maeda Nobuyo , Tsai Yau-Sheng
Published: March 2022

PPARγ, a nuclear receptor that regulates adipocyte development and glucose homeostasis, is the molecular target of a class of insulin-sensitizing drugs. Loss-of-function mutations in PPARG gene are implicated in human hypertension and PPARγ agonists decrease blood pressure both in animal models and human patients. We have previously generated mice with varying Pparg expression created by modifying its 3′-UTR sequence, and concluded an inverse relationship between PPARγ expression level and blood pressure. Interestingly, mice with extremely low PPARγ expression exhibited an increase in systolic blood pressure without significant change in diastolic blood pressure, ultimately causing a significant increase in pulse pressure. Thus, we hypothesized that PPARγ deficiency impairs aortic elasticity, resulting in the increase in pulse pressure. Aortic pulse wave velocity (PWV) was significantly increased in PPARγ hypomorphic mice, suggesting that aortas of PPARγ hypomorphic mice were stiffer than those of wild-type mice. The measurement of mechanical property by atomic force microscope (AFM) showed higher tissue rigidity in the aorta of PPARγ hypomorphic mice. Expression of elastin in aortas was significantly decreased, and negatively correlated with aortic PWV in PPARγ hypomorphic mice. This was accompanied by increased elastin fiber breaks, elastolytic activity, and expressions of MMP-9 in aortas of PPARγ hypomorphic mice. The elevated PWV in PPARγ hypomorphic mice was ameliorated after treatment of PPARγ agonist. Together, our results suggest that PPARγ deficiency impairs elasticity in aorta and underscore the importance of PPARγ in the regulation of blood pressure and vascular reactivity.
Abstract 208: Laminar Shear Stress Decreases Endothelial Microparticles Release by a Nitric Oxide- and ABCA1-Dependent Mechanism
Authors: Vion Anne C , Ramkhelawon Bhama , Loyer Xavier , Chironi Gilles , Tedgui Alain , Lehoux Stephanie , Boulanger Chantal M
Published: March 2022

Microparticles (MP) are shed-membrane sub-micron vesicles released upon cell activation or apoptosis. Their formation is associated with loss of cell membrane asymmetry and exposure of phosphatidylserine (PS), resulting from increases in floppase activities such as ABCA1. Plasma endothelial MP (EMP) are surrogate markers of endothelial dysfunction and their levels increase in patients with cardiovascular diseases. However, the mechanisms governing EMP release in vivo remain largely unknown. As nitric oxide (NO) is a key regulator of the vasoprotective effect of laminar shear stress (SS) on the endothelium, we tested the hypothesis that endothelial NO regulates EMP release under different SS profiles.

Shed-annexinV+ EMPs were analyzed by flow cytometry in the medium of confluent HUVECs (passage 1-3) subjected to either high- (15 dyne/cm2; atheroprotective) or low- (1.5 dyne/cm2; atheroprone) SS conditions for 24 hrs. HUVECs exposed to high-SS released 3-fold less EMPs compared to low-SS conditions (326±37 vs. 130±23AnnV+MPs/μL, p<0.001). High-SS EMPs also externalized less PS, as estimated by the mean fluorescence of FITC-AnnV labelling (15.6±6.1 vs. 24.2±9.4 AU, p<0.001). Exposure to L-NAME (10-5M), but not D-NAME, increased high-SS-induced EMP release by over 3-fold (p<0.001) and the NO donor SNAP (10-5M) significantly decreased low-SS-induced EMP formation by 50% (p=0.05). In addition, L-NAME increased the mean fluorescence of EMP labelling under high SS (p<0.05), whereas SNAP decreased it under low SS (p<0.05). We then evaluated ABCA1 expression under these conditions. high-SS significantly decreased the expression of ABCA1 both at the mRNA and protein level by 2- and 20-fold, respectively (p<0.05 vs low SS). L-NAME augmented ABCA1 mRNA level in cells exposed to high SS (p<0.05), whereas SNAP significantly decreased ABCA1 mRNA level under low SS conditions (p<0.05). Finally, the LXRα agonist TO90131 (0.1mM) not only increased endothelial ABCA1 expression by 20-fold, but also augmented MP level under high SS conditions.

Altogether, these results demonstrate that atheroprotective shear stress limits the release of procoagulant endothelial MP through a NO-dependent regulation of endothelial ABCA1 expression.
Abstract 209: Inhibition of AMP-Activated Protein Kinase Accentuates Lipopolysaccharides-Induced Lung Endothelial Barrier Dysfunction and Lung Injury in Vivo
Authors: Xing Junjie , Viollet Benoit , Wang Qilong , Zou Ming-Hui
Published: March 2022

AMP-activated protein kinase (AMPK) is reported to have vascular protective effects, but whether AMPK regulates endothelial barrier functions and protects lung function remains unknown. The aim of this study was to determine the roles of AMPK in lipopolysaccharide (LPS)-induced lung endothelial barrier dysfunction (
in vitro
and
in vivo
) and lung injury (
in vivo
). Cultured human pulmonary artery endothelial cells (HPAECs) and AMPKα subunit-deficient mice and wild-type (WT) control (C57BL/6J) mice were used to study the molecular mechanisms by which LPS induced lung endothelial barrier dysfunction and lung injury. In cultured HPAECs, LPS markedly increased endothelial hyperpermeabilty in parallel with a marked decrease in both AMPK phosphorylation at Thr172 and AMPK activity. Consistent with this latter observation, AMPK activation with AICAR treatment attenuated LPS-induced endothelial hyperpermeabilty
in vitro
. Intratracheal administration of LPS (1 mg/kg) in WT C57BL6 mice markedly reduced AMPK phosphorylation at Thr172 in lung tissue extracts, but markedly increased protein contents and cell numbers in bronchial aveolar lavage fluid and Evans blue dye infiltration in the lung
in vivo
. Pretreatment with AICAR, a potent AMPK activator, markedly reduced LPS-enhanced protein contents, cell counts, and inflammatory cell infiltration into alveolae. These lung injury indicators were significantly enhanced in AMPK-knockout mice compared to WT C57BL6 mice, supporting that AMPK inhibition by LPS accentuated lung injury. Mechanistically, we found that AMPK activation with AICAR attenuated LPS-induced endothelial hyperpermeabilty by activating the Rac/Cdc42/PAK pathway with concomitant inhibition of the Rho pathway, and decreased VE-cadherin phosphorylation at Tyr
658
. We conclude that (1) LPS inhibits AMPK; (2) AMPK inhibition accelerates LPS-induced endothelial barrier dysfunction and lung injury; and (3) AMPK activation attenuates LPS-induced pulmonary vascular hyperpermeability and lung injury.
Abstract 214: Engagement of Platelet Toll-like Receptor 9 by Classical and Novel Endogenous Ligands Promotes Platelet Hyperreactivity and Thrombosis
Authors: Panigrahi Soumya , Ma Yi , Hong Li , Gao Detao , Salomon Robert G , Byzova Tatiana V , Podrez Eugene A
Published: March 2022

A prothrombotic state and increased platelet reactivity are common in pathophysiological conditions associated with oxidative stress and infections. Such conditions are associated with an appearance in circulation of non-self or altered-self ligands that are commonly recognized by toll-like receptors (TLR). Platelets express a number of TLR, including TLR9, however, the role of TLR in platelet function and thrombosis is poorly understood. In this study, we demonstrate that carboxyalkylpyrrole protein adducts (CAPs), an altered-self ligand generated in oxidative stress, promotes platelet activation, granule secretion, and aggregation in vitro and thrombosis in vivo via the TLR9/MyD88 pathway. We demonstrate that CAPs represent a novel unconventional ligand for TLR9. Furthermore, we demonstrate that unmethylated CpG sequences, a canonical non-self ligand of TLR9, induce strong platelet activation via TLR9/MyD88. Platelet activation by TLR9 ligands induces IRAK1 and AKT phosphorylation, and is Src kinase dependent. Physiological platelet agonists act synergistically with TLR9 ligands by inducing TLR9 expression on the platelet surface. Thus, our study demonstrates that platelet TLR9 links oxidative stress, innate immunity, and thrombosis by serving as a sensor of canonical non-self and unconventional altered-self ligands.
Abstract 216: Cyclophilin A Regulates Hemostasis by Affecting Multiple Platelet Functions
Authors: Wang Lian , Baskaran Padmamalini , Sowden Mark , Morrell Craig N , Berk Bradford C
Published: March 2022

Background:
Cyclophilin A (CyPA) is ubiquitously expressed and regulates multiple cellular functions including protein folding, intracellular trafficking and signal transduction. Extracellular CyPA is an inflammatory mediator that contributes to atherosclerosis, aortic aneurysms, vascular remodeling and myocardial ischemic reperfusion injury. Recent studies have shown that interactions among platelets, endothelial cells and leukocytes play important roles in inflammation. However, the role of CyPA in platelet function and thrombosis remains unclear. We hypothesized that CyPA is involved in platelet activation, resulting in an impact on hemostasis and arterial thrombosis.

Methods:
Hemostasis was assessed by determining tail bleeding times in CyPA
-/-
mice (28±3d of age) compared to age matched wild-type (WT) littermates. Blood loss during the bleeding time assays was quantified by determination of hemoglobin concentration as absorbance at 575nm. Blood was collected in EDTA tubes from the retro-orbital plexus and platelet counts were measured using an automatic hemocytometer. Flow cytometric analysis was used to determine the expression of P-selectin and fibrinogen binding in response to various agonists. Washed platelets from WT and CyPA
-/-
mice were allowed to spread on immobilized fibrinogen and visualized by differential interference contrast (DIC) microscopy.

Results:
Tail bleeding times were significantly prolonged in CyPA
-/-
mice (n=13) compared to WT mice (n=20) (median bleeding time: 1025 s vs. 456 s). Accordingly, blood loss during the bleeding time assay was also significantly increased in CyPA
-/-
mice (OD575: 0.54±0.22 vs. 0.08±0.01). CyPA
-/-
mice have similar platelet counts as the age matched WT mice. CyPA deficiency impaired P-selectin expression in response to thrombin and ADP plus
U46619
, and fibrinogen binding in response to thrombin. Furthermore, CyPA
-/-
platelets had decreased filopodia and lamellipodia formation on immobilized fibrinogen. CyPA
-/-
platelets exhibited a decrease of 41% in spreading area as compared to WT platelets.

Conclusion:
These data indicate for the first time that CyPA regulates hemostasis through affecting multiple platelet functions including α-granule secretion, platelet activation and spreading.
Abstract 217: The P2Y12 Antagonists, 2MeSAMP and AR-C69931MX, Inhibit Platelet Activation Through the P2Y12/Gi-Dependent Mechanism
Authors: Xiang Binggang , Zhang Guoying , Gartner T Kent , Smyth Susan S , Li Zhenyu
Published: March 2022

Objective—
ADP is an important physiological agonist that induces integrin activation and platelet aggregation through it receptors the P2Y
1
(G
q
-coupled) and P2Y
12
(G
i
-coupled). P2Y
12
plays a critical role for platelet activation and thrombosis. Adenosine-based P2Y
12
antagonists, 2-methylthioadenosine 5
’
-monophosphate triethylammonium salt (2MeSAMP) and AR-C69931MX are widely used to demonstrate the role of P2Y
12
. AR-C69931MX (Cangrelor) is also under clinical trials for thrombotic diseases. However, a recent study reported that both 2MeSAMP and AR-C69931MX raise intra-platelet cAMP levels and inhibit aggregation through a P2Y
12
-independent mechanism. The present work, by using P2Y
12
deficient mice, sought to clarify previous conflicting reports and to elucidate the mechanisms by which 2MeSAMP and AR-C69931MX inhibit platelet activation and thrombosis.

Methods and Results—
We show that 2MeSAMP and AR-C69931MX inhibited aggregation and ATP release in platelets from wild type but not in P2Y
12
deficient mice. 2MeSAMP and AR-C69931MX neither raised intracellular cAMP levels nor induced VASP phosphorylation in both mouse and human platelets. Furthermore, unlike the activators (PGI
2
and forskolin) of the cAMP pathway, 2MeSAMP and AR-C69931MX failed to inhibit platelet aggregation, calcium mobilization, Akt phosphorylation, and Rap1b activation in P2Y
12
deficient platelets. In addition, while injection of AR-C69931MX inhibited thrombus formation in a FeCl
3
-induced thrombosis model in wild type mice, it failed to affect thrombus formation in P2Y
12
deficient mice.

Conclusion—
These data together demonstrate that 2MeSAMP and AR-C69931MX inhibit platelet function through the P2Y
12
-dependent mechanism both
in vitro
and
in vivo
.
Abstract 24: MicroRNA-27b Is a Regulatory Hub in Lipid Metabolism and Is Altered in Dyslipidemia
Authors: Vickers Kasey C , Shoucri Bassem M , Levin Michael G , Wu Han , Osei-Hwedieh David , Pearson Daniel S , Collins Francis S , Sethupathy Praveen , Remaley Alan T
Published: March 2022

Cellular and plasma lipid levels are tightly controlled by complex gene regulatory mechanisms. MicroRNAs (miRNAs) are post-transcriptional regulators of gene expression and have emerged as important modulators of lipid homeostasis, but the extent of their role has not been systematically investigated. In this study, we performed high-throughput small RNA sequencing and detected approximately n=150 miRNAs in mouse liver. We then employed an unbiased,
in silico
strategy to identify miRNA regulatory hubs in lipid metabolism, and miR-27b was identified as the strongest such hub in human and mouse liver. In addition, hepatic miR-27b levels were determined to be sensitive to plasma hyperlipidemia, as evidenced by its 3-fold up-regulation in the liver of mice on a high-fat diet (42% calories from fat). Further, we showed in a human hepatocyte cell line (Huh7) that miR-27b regulates the expression (mRNA and protein) of several key lipid-metabolism genes. Finally, hepatic levels of miR-27b and its target genes, which include
ANGPTL3
and
GPAM
, were inversely regulated in a mouse model of severe dyslipidemia and atherosclerosis. Conclusion: miR-27b is responsive to lipid levels, and acts as a “regulatory hub” in lipid metabolism, controlling multiple genes involved in dyslipidemia.
Abstract 250: Increased 18F-FDG Uptake in the Aortic Wall of β-Aminopropionitrile Exposed Rats May Be Predictive of AAA Rupture
Authors: English Sean J , Piert Morand R , Diaz Jose A , Ghosh Abhijit , Sherman Phillip S , Quesada Carole A , Pechota Angela , Eliason Jonathan L , Henke Peter K , Upchurch Gilbert R
Published: March 2022

Objectives:
Ruptured abdominal aortic aneurysm (AAA) is associated with a high mortality rate, and a noninvasive test is necessary to detect vascular wall inflammation that may aid in predicting AAA rupture. The ability of 18F-FDG positron emission tomography (PET) imaging to identify increased inflammation in the porcine pancreatic elastase (PPE) perfusion model in Sprague-Dawley rats exposed to β-aminopropionitrile (BAPN) to stimulate AAA rupture was evaluated.

Methods:
Starting 3d prior to infrarenal abdominal aortic exposure to PPE, BAPN (300mg/kg subcutaneous) was administered daily until AAA harvest or rupture (BAPN, n=9). Control rats only underwent PPE exposure (n=8). Aortic diameter was evaluated at 3, 7, and 14d post PPE exposure utilizing ultrasound (US, 12MHz). Both groups were imaged with 18F-FDG microPET (control n=4, rupture n=3) 7d post PPE exposure. Maximum AAA to mean muscle 18F-FDG uptake ratios (AMR) were determined. Matrix metalloproteinase (MMP) 2 and 9 zymogramatic activities, in terms of integrated optical density (IOD), were assessed at 7d (control n=3, BAPN n=4) and 14d post PPE exposure (control n=3, rupture n=3).

Results:
Mean 7d aortic diameter increases of 247% and 398% (p=0.014) were observed in control and rupture groups, respectively, with no evidence of dissection in BAPN exposed rats on US. Three BAPN exposed rats ruptured within 24h after 18F-FDG microPET, with mean AMRs for the control and ruptured groups of 86.5 and 281.9 (p=0.048), respectively. IOD values of 4655 and 8373 (p=0.140) were determined for MMP9 activity in 14d control and ruptured AAA tissue, respectively, representing an 80% increase in ruptured AAA tissue. IOD values of 4760 and 1190 (p=0.032) were determined for MMP2 activity in 14d control and ruptured AAA tissue, respectively, representing a 75% decrease in ruptured AAA tissue. No differences in MMP2 or 9 activities for control and BAPN exposed rats were observed at 7d post PPE exposure.

Conclusions:
Alterations in MMP2 and 9 activities in BAPN exposed, ruptured, rat AAA tissues parallel alterations in MMP2 and 9 activities in ruptured, human AAA tissues observed by others. Increased 18F-FDG uptake by rat AAAs that subsequently ruptured suggests that FDG-PET may be used to predict AAA rupture in humans.
Abstract 252: Potently Proteolytic Microvesicles Derived from Neutrophils Are Present Within the Intraluminal Thrombus of Human Abdominal Aortic Aneurysms
Authors: Folkesson Maggie , Li Chunjun , Swedenborg Jesper , Roy Joy , Williams Kevin Jon , Eriksson Per , Liu Ming-Lin
Published: March 2022

BACKGROUND—
Extracellular matrix degradation and the formation of a non-occlusive intraluminal thrombus (ILT) are important clinical features of human abdominal aortic aneurysms (AAAs). The aortic wall under the ILT is thinner with less elastin than the wall without ILT. Leukocytes are trapped within the ILT, mainly in its luminal layer, and proteolytic activity derived from these cells could contribute to degradation of the underlying wall. In prior work, however, we detected only scant enzymatic activity of soluble, secreted matrix metalloproteinases (MMPs) in the abluminal layer of the ILT, i.e., the layer closest to the vessel wall. AIM- To examine two major insoluble, transmembrane MMPs - namely, ADAM17 (TACE) and ADAM10, in the abluminal layer of the ILT as a source of proteolytic activity.

METHODS/RESULTS—
Aortic segments and ILTs were collected from 9 male and 3 female patients, all present or former smokers, during elective AAA surgery. Immunoblots showed abundant ADAM17 and ADAM10 in all three layers (luminal, middle, and abluminal) of their ILTs. ADAM 17 exhibited high levels of proteolytic activity in all layers of the ILT. By immunohistochemical staining, ADAM10 co-localized with the neutrophil marker CD66acd, but not with the macrophage marker CD163. Importantly, ADAM10 and CD66acd co-localized within the abluminal ILT in structures < 2 μm, without intact cells or nuclei. FACS analysis confirmed the presence of a significant number of microvesicles (MVs) in the abluminal layer of ILT. Moreover, exposure of cultured human HL-60 neutrophils to tobacco smoke stimulated large, time-dependent increases in the cellular content of both ADAM17 and ADAM10, cleavage of these proteases into their active forms, and release of enzymatically active ADAM17- and 10-positive MVs.

CONCLUSIONS—
These results for the first time implicate persistent, enzymatically active transmembrane ADAM proteases within cell-free structures in the abluminal ILT, adjacent to the aortic wall of human AAA, as novel participants in mural weakening. Moreover, the production of proteolytically active MVs from smoke-exposed neutrophils provides a novel molecular mechanism for the vastly accelerated risk of AAA in smokers.
Abstract 254: Cellular Heterogeneity of the Ascending Aorta Relationship to AngII-Induced Medial Hemorrhage
Authors: Jung Kyung Sik , Rateri Debra L , Howatt Deborah A , Balakrishnan Anju , Moorleghen Jessica J , Daugherty Alan
Published: March 2022

Introduction and Objective:
Infusion of angiotensin II (AngII) into mice generates pronounced aneurysms that are localized to the ascending aorta. Following prolonged infusion of AngII, the resulting ascending aneurysms are characterized by pronounced lumen dilation and concentric medial thickening with progressive expansion between elastin layers from the lumen to the adventitia of the aorta. The presence of medial hemorrhage is a common characteristic within 5 days of initiating AngII infusion. The purpose of this study was to determine whether the location of medial hemorrhage coincided with overt structural or cellular transition within the aorta.

Methods and Results:
Eight-week-old, male, C57BL/6 mice were fed a normal laboratory diet and infused with either saline (N=5) or AngII (1,000 ng/kg/min, N=20) subcutaneously using osmotic mini pumps for 5 days. Mice were terminated after 5 days of AngII infusion. Thirty percent (6 out of 20) displayed hemorrhage, visible from the ascending aorta to the aortic arch. Serial sections (10 μm) were acquired through the entire aortic root and ascending aorta. Alpha-actin and reticular fibroblast (ER-TR7) antibodies detected the location of smooth muscle cells (SMC) and fibroblasts, respectively. In both groups, there was a transition within the aortic root in which the media no longer immunostained positively for alpha actin. Conversely, this region immunostained positively for fibroblasts. Hemorrhages occurred distal to the cellular phenotypic changes seen in the aortic root.

Conclusion:
These findings suggest that the development of AngII-induced medial hemorrhage within the ascending aorta did not correspond to the medial cellular transitions present in the aortic root.
Abstract 259: Generation of Model HDL Complexes to Dissect the Role of Apolipoprotein A-II on Antioxidative and Anti-inflammatory Properties of HDL
Authors: Gauthamadasa Kekulawalage , Urbancic Michelle , Basford Joshua E , Homan Reyn , Jerome W Gray , Hui David Y , Silva R A Gangani D
Published: March 2022

Human high density lipoproteins (HDL) are divided into two major subpopulations based on lipoprotein composition. The predominant subpopulation contains both major apolipoproteins apoA-I and apoA-II (termed LpA-I/A-II), while the minor subpopulation contains only apoA-I (termed LpA-I). It is speculated that LpA-I/A-II could demonstrate different anti-oxidative and anti-inflammatory properties compared to LpA-I. The goal of the current work was to dissect out any contributions from apoA-II on such beneficial functions of HDL. We generated a series of reconstituted (r)LpA-I/A-II in which major apolipoprotein ratios mimic native LpA-I/A-II (apoA-I: apoA-II; 2:1, 2:2 and 2:3) but lack the anti-oxidative enzymes in native HDL. First, we generated the precursor rLpA-I by reacting discoidal HDL with recombinant lecithin cholesterol acyl transferase (LCAT) in the presence of low density lipoproteins. The rLpA-I consist of a single population of ∼93 Å particles. Cross-linking followed by MALDI-MS indicated exactly three apoA-I per rLpA-I. Different rLpA-I/A-II complexes were generated by adding the required amount of apoA-II into rLpA-I followed by isolation of rLpA-I:A-II using size exclusion chromatography. All rLpA-I/A-II showed comparable particle diameter to rLpA-I. Electron microscopy clearly captured the spherical nature of these particles and agarose gel electrophoresis demonstrated the presence of a neutral lipid core in rLpA-I and rLpA-I/A-II compared to discoidal HDL. All particles contain comparable amounts of total protein 36-40%, phospholipids 40-45%, cholesteryl esters 17-22%, free cholesterol <1% and triglycerides <1%. In vitro anti-oxidative functional assays utilizing copper-mediated oxidation demonstrated ∼20% better anti-oxidative capacity for rLpA-I/A-II (apoA-I: apoA-II, 2:1) compared to rLpA-I. We are currently in the process of testing anti-oxidative properties of other rLpA-I/A-II complexes. Experiments are currently underway to assess differences of anti-inflammatory properties of these complexes by quantitative measurements of adhesion molecule VCAM, ICAM and E-selection using flow cytometry in cell culture.
Abstract 283: Novel Mechanism of Low-Density Lipoprotein--Derived Cholesteryl Ester Uptake and Hydrolysis During Macrophage Foam Cell Formation
Authors: Meyer Jason M , Ji Ailing , Cai Lei , van der Westhuyzen Deneys R
Published: March 2022

Macrophage foam cells are a defining pathologic feature of atherosclerotic lesions. Classically, scavenger receptor-mediated uptake of oxidized or enzyme-modified LDL is cited as a mechanism for foam cell formation. Recent studies have demonstrated that at high concentrations, native LDL can also induce macrophage lipid accumulation. LDL particles are taken up by macrophages as part of bulk fluid pinocytosis. However, the uptake and metabolism of cholesterol derived from native LDL during macrophage foam cell formation has not been clearly defined. Previous studies suggested that selective CE uptake might contribute to cholesterol uptake independently of LDL endocytosis. In this study we demonstrate that the majority of cholesterol taken up from LDL is acquired by selective CE uptake and not by pinocytosis and LDL degradation. CE uptake does not saturate at high LDL concentrations and is not down-regulated during cholesterol accumulation. LDL modification and scavenger receptors do not appear to play a major role in macrophage selective CE uptake. Following uptake from LDL, CE is rapidly hydrolyzed in macrophages by a novel chloroquine- and temperature-sensitive pathway. Our findings suggest that selective CE uptake plays a major role in macrophage cholesterol homeostasis during foam cell formation.
Abstract 29: Fate-Tracing Demonstrates that Medial SMCs, but Not Mature ECs, Contribute to Neointimal Cell Population in Injured Arteries
Authors: Hong Michael , Gupta Tushar , Fu Chunhua , Berceli Scott , Jiang Zhihua
Published: March 2022

It has been proposed that various groups of cells, such as medial SMCs (mSMCs), bone marrow progenitors, and mature ECs (mECs) hold the potential to become neointimal SMCs via phenotypic switching, differentiation, or endothelial-mesenchymal-transition (EMT). However, no definitive evidence has been provided to support these claims, which renders the in vivo significance of these events in question.

Using an inducible Cre-loxP system, where the production of a reporter (e.g. β-gal) is driven by a highly definitive marker gene for mSMCs (e.g. SMC myosin heavy chain, SMMHC) or mECs (e.g. stem cell leukemia, SCL), we traced the fate of these cells during neointimal hyperplasia (NIH) in ligated common carotid and wire-injured femoral arteries. Tamoxifen induction resulted in a homogeneous labeling of mSMCs of the unmanipulated arteries of R26R;SMMHC-CreERtm mice. Following arterial injury, SMCs that were initially located in the medial layer presented in neointimal lesions; however, the fraction of these cells varied widely among vessels with either type of injury. This heterogeneity was not secondary to the random loss of the genetic label as evidenced by the stable β-gal activity in SMC culture. Addition of a second (4wks after surgery) induction did not change the pattern and fraction of the labeled SMCs in the neointima, indicating that few recruited progenitors transform into mature SMCs. This observation is further supported through examination of mice chimeric for R26R;SMMHC-CreERtm bone marrow, where β-gal activity was not detected in injured arteries though strongly induced in culture following PDGF treatment. Neointima formed in R26R;SCL-CreERtm arteries demonstrated a co-localization of β-gal activity to the repopulated ECs, with none being detected in cells underneath the EC monolayer, suggesting a role for mECs in re-endothelialization and indicating EMT as a rare event in NIH development.

We have provided direct evidence for the contribution of mSMCs to NIH. The recruited vascular progenitors, particularly bone marrow cells, do not differentiate into fully mature SMCs in NIH lesion. EMT does not serve as a mechanism for populating neointimal cells following vascular injury while occurring under other vascular disease conditions.
Abstract 291: Health Effects of Northern Contaminants and Alcohol Consumption in a Rodent Model of Human Metabolic and Cardiovascular Diseases
Authors: Florian Maria , Yan Jin , Petrov Ivan , Caldwell Don , Coughlan Melanie C , Laziyan Mahemuti , Li Nanqin , Willmore William , Jin Xiaolei (Dawn)
Published: March 2022

For the last few decades, Northern populations have been exposed to elevated levels of Northern contaminants (NC) such as polychlorinated biphenyls and heavy metals, mainly through consumption of contaminated fish and marine mammals. Along with this, there has also been an increased prevalence of obesity, hypertension, diabetes, and cardiovascular disease. Although changes in lifestyle and diets have been associated with this prevalence of chronic diseases, it remains to be clarified if exposure to NC also plays a part, and if lifestyles choices such as consumption of alcohol may modulate the effects of NC.

This study explored these questions using a rodent model of human metabolic and cardiovascular diseases.

Obese male JCR rats at 8 weeks of age were acclimatized on a purified AIN93G diet. Animals were treated with 10% alcohol in drinking water or drinking water only for 6 weeks. From the third week of alcohol treatment, animals were orally dosed with a mixture of 23 NC (NCM) at 0 (corn oil), 1.6, or 16 mg/kg BW/day for 4 weeks. During the whole study, body weight and water/alcohol consumption were measured daily. Food consumption was measured weekly. Blood and organs were collected and weighed at the end of the study. Tissue samples were analyzed for hematology and serum NCMs, glucose, insulin, and ethanol levels, as well as clinical biochemistry markers.

Serum NCM levels increased with NCM dose, and were mostly in the range of NCM levels found in Inuit blood. Serum NCM levels were generally lower in the rats treated with alcohol than those without. NCM at 16 mg/kg BW slightly decreased body weight gain and water and food consumption especially in the rats given no alcohol. Regardless of alcohol, this dose of NCM significantly increased absolute and relative liver weight, relative kidney weight, and serum C-reactive protein and ethanol levels. NCM also decreased serum paraoxonase-1, insulin, cholesterol, triglyceride, HDL, and LDL levels.

Our data suggested that NCM at levels relevant to human exposure may alter lipid metabolism and/or transport in the liver, insulin secretion from the pancreas, and circulating cardiovascular health markers.

This research was supported by Canadian Institute of Health Research, Postdoctoral Fellowship No 200910-JHF-214255.
Abstract 318: Hepatic Site-1 Protease Knockdown in Mice Decreases Proprotein Convertase Subtilisin/kexin Type 9 Expression
Authors: Basu Debapriya , Lei Xia , Costet Philippe , Jin Weijun
Published: March 2022

Introduction—
Site-1 protease (S1P) is a proprotein convertase involved in activation of transcription factors from their membrane-bound precursors in a process known as Regulated Intramembrane Proteolysis. These factors include Sterol Regulatory Element Binding Proteins (SREBPs) and CRE Binding Protein-H. SREBPs are the transcriptional master regulators of
de novo
cholesterol and fatty acid biosynthetic genes, and also positively regulate Low density lipoprotein receptor (LDLR) and Proprotein convertase subtilisin/kexin type 9 (PCSK9) expression. Inhibition of PCSK9 is a promising drug targeting strategy for treatment of dyslipidemia since the major function of PCSK9 is to degrade LDLR.

Hypothesis—
S1P controls PCSK9 expression and function in the liver.

Results—
Inducing Cre-LoxP mediated DNA recombination in the liver of S1P flox mice by three different approaches, we consistently observed that S1P knockdown significantly decreases PCSK9 mRNA expression in the liver and reduces plasma PCSK9 concentrations compared to S1P flox control mice. This effect is largely due to a decrease in PCSK9 mRNA synthesis without a change in its mRNA stability. The reduction of PCSK9 mRNA correlates with downregulation of the SREBP pathways and has a positive effect on hepatic LDLR protein levels as evident in mice after food entrainment. Also, we found that S1P differentially regulates PCSK9 and LDLR mRNA levels in a dose- and time-dependent manner. Finally a constitutively active form of SREBP, but not CREBH, almost restores PCSK9 expression in isolated hepatocytes from S1P knockdown mice to that of S1P flox control. Hence, S1P regulates PCSK9 expression through SREBPs.

Conclusion—
Taken together, our study reveals the physiological role of liver S1P in regulating hepatic and plasma PCSK9 levels and influencing its function. This may provide a novel strategy to inhibit PCSK9.
Abstract 340: Phenotypic Conversion of Perivascular Adipose Tissue in Uninephrectomized Apoe-Deficient Mice: Possible Role in the Early Stage of Atherosclerosis in Chronic Kidney Disease
Authors: Kawahito Hiroyuki , Yamada Hiroyuki , Kishida Sou , Kato Taku , Irie Daisuke , Ikeda Kouji , Okigaki Mitsuhiko , Mori Yasukiyo , Matsubara Hiroaki
Published: March 2022

BACKGROUND:
Chronic kidney disease (CKD) is now recognized to be an independent risk factor for cardiovascular disease. Recently, perivascular adipose tissue (PVAT) has been shown to play a crucial role in the pathogenesis of atherosclerosis; however, the role of PVAT in the CKD-associated proatherogenic actions remains undefined.

METHOD AND RESULT:
Eight-week-old apoE deficient mice underwent uninephrectomy (UNX) or sham operation and were fed a western diet starting at 12 wks of age. UNX mice at the age of 20 wks showed a significant exaggeration of atherosclerotic lesion area (53%, P<0.05). Hemodynamic data, lipid profile, and plasma creatinine concentrations did not differ between the two groups. To elucidate the underlying mechanisms of minor renal insufficiency-induced atherosclerosis, we first examined the number of circulating inflammatory monocytes and the mRNA expression levels of VCAM-1 and ICAM-1 in the vasculature at 16 wks of age. However, there was no difference between the two groups, suggesting that uninephrectomy did not effect on either western diet-induced mobilization of bone marrow-derived monocytes or endothelial activation in the early stage of atherosclerosis. We therefore focused on the PVAT surrounding the thoracic aorta, which showed multilocular fat droplet and high expression levels of brown adipocyte-specific genes. The fat pad weight and cross-sectional area of PVAT including the infiltration of CD45+ cells did not differ between the two groups. While mRNA expression levels of adipocyte differentiation marker genes (PPARγ, FABP4, and adiponectin) were equivalent between the two groups, brown adipocyte-specific marker genes (UCP-1, Elovl3) showed a significantly lower expression in UNX mice (P<0.01). In contrast, white adipocyte-specific marker gene Tcf21 was markedly up-regulated (101%, P<0.05), accompanied by the increased expression of angiotensinogen gene (85%, P<0.05). The concentration of serum angiotensin II did not differ between the two groups.

CONCLUSION:
Phenotypic conversion of perivascular brown to white adipose tissue along with the depot-specific local activation of renin-angiotensin system is a possible mechanism underlying the early stage of atherosclerosis in CKD.
Abstract 35: Atheroprotective Effects of Adiponectin Overexpression in a Model of AngII-Accelerated Atherosclerosis
Authors: van Stijn Caroline M , Kim Jason , Becerra Diana , Tangirala Rajendra K
Published: March 2022

Adiponectin, an adipocytokine derived from adipose tissue, exerts anti-inflammatory and anti-atherogenic effects on vascular cells. Although low plasma adiponectin levels are associated with the metabolic syndrome, diabetes and cardiovascular disease, it is unknown whether elevation of adiponectin levels protects against angiotensin II (AngII)-mediated vascular inflammation and accelerated atherosclerosis. Here, we tested the hypothesis that increasing plasma adiponectin levels provides therapeutic benefit by inhibiting vascular actions of AngII promoting vascular inflammation and accelerated atherosclerosis. Low-density lipoprotein receptor-deficient (LDLR-/-) mice fed high-fat and infused with Ang-II were injected adenovirus expressing GFP (Ad-GFP) or mouse adiponectin (Ad-APN). After 8 weeks, plasma adiponectin levels were 5-fold higher in Ad-APN than Ad-GFP. This was accompanied by significant elevation of plasma HDL in Ad-APN mice (24% elevation vs ad-GFP). Quantification of atherosclerosis revealed a significant inhibition of atherosclerosis in Ad-APN (45% reduction vs Ad-GFP). Gene expression analyses revealed that adiponectin substantially inhibited mRNA levels of inflammatory and atherogenic genes (CD68, ICAM-1, AT-1R, osteopontin, MCP-1 and CCR2, scavenger receptors, SRA-1 and CD36) in the aortic wall. Interestingly, adiponectin increased expression of anti-inflammatory cytokine, 1L-10 and the reverse cholesterol transport genes, ABCA1 and ABCG1 in the aortic wall. Furthermore, adiponectin increased hepatic ABCA1 and PPAR-alpha expression. These data strongly support the concept that increasing plasma adiponectin levels is an effective therapeutic strategy to inhibit AngII-mediated vascular inflammation and accelerated atherosclerosis.
Abstract 358: Cyclic Strain-Induced Release of Thrombomodulin from Human Aortic Endothelial Cells
Authors: Martin Fiona A
Published: March 2022

Introduction:
Loss of thrombomodulin (TM), a cofactor in the protein C anti-coagulant pathway, through a combination of reduced TM expression and elevated TM release, is a key feature of endothelial dysfunction leading to vascular diseases. Reduction in endothelial TM levels for example is viewed as a major cause of cyclic strain-induced early vein graft failure. In this respect, our understanding of the mechanistic relationship between vessel stretch and TM expression/release in endothelial cells is very limited, with a particular shortage of in vitro models. In this study, we investigated how cyclic circumferential strain negatively regulates thrombomodulin levels in human aortic endothelial cells (HAECs) in vitro.

Methods:
A Flexercell® Tension-Plus™ FX-4000T™ system (Flexcell International Corp., Hillsborough, NC) was routinely employed to subject HAECs to equibiaxial cyclic strain over a range of doses (0, 2.5 and 7.5% stretch; 60 cycles/min; cardiac waveform) and times (0-48 hours). TM expression was analyzed via Western blotting and qRT-PCR, whilst TM release was monitored by ELISA.

Results:
(i) Cyclic strain of HAECs led to significant force- and time-dependent reductions in TM expression (mRNA and protein) in conjunction with elevated TM release into media; (ii) Inclusion of either GM6001 (25 μM) or apocynin (10 mM) into strain experiments to block MMP-2/9 and NADPH oxidase activities, respectively, had no effect on strain-induced TM release; (iii) Control studies demonstrated that treatment of HAECs with neither TNF-α (0, 10, 100 ng/ml) nor oxLDL (10, 50, 145.7 μg/ml) increased TM release from HAECs - however, oxLDL treatment was found to dose-dependently potentiate cyclic-strain-induced TM release.

Conclusions:
Cyclic strain negatively regulates TM levels in endothelial cells in vitro. Moreover, release of TM from HAECs does not appear to involve the strain-dependent induction of either MMP-2/9 proteolytic activity or NADPH oxidase-dependent superoxide production. Finally, the cyclic strain-induced release of TM appears to be potentiated by inflammatory atherogenic conditions such as high oxLDL levels.