bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 400: Protective Effects of CCR2 Deficiency on Ascending Aortic Aneurysm Formation Are Not Mediated via MCP-1
Authors: Rateri Debra L , Howatt Deborah A , Balakrishnan Anju , Moorleghen Jessica J , Cassis Lisa A , Daugherty Alan
Published: March 2022

Background and Objective: Infusion of AngII into hyper- and normocholesterolemic mice leads to formation of aneurysms in the ascending aorta. Aneurysms in this region have markedly different pathology from those formed in the abdominal aorta. We have demonstrated previously that deficiency of the chemokine receptor, CCR2, attenuated ascending aortic aneurysm formation in hypercholesterolemic mice. The purpose of this study was to define whether deficiency of CCR2 also attenuated AngII-induced ascending aortic aneurysms in normolipidemic mice. Furthermore, we also determined whether the effects were attributable to its primary ligand, MCP-1.

Methods and results:
Male C57BL/6, CCR2
-/-
and MCP-1
-/-
mice (The Jackson Laboratory, Bar Harbor, ME) were fed a normal diet. Mice were infused with either saline or AngII (1,000 ng/kg/min; N=20/group) subcutaneously via osmotic mini-pump for 28 days. Systolic blood pressure (SBP) was measured by a tail cuff-based technique. AngII increased SBP significantly and equivalently in all genotypes relative to saline-infused mice (P<0.05). Serum MCP-1 concentration was measured by ELISA. As expected, MCP1
-/-
mice had no measurable MCP-1; while CCR2
-/-
mice had a 10 fold increase compared to C57BL/6 mice (P<0.001). En face arch and ascending aortic areas were measured. AngII infusions significantly increased aortic areas compared to saline infused mice (P<0.001) in all genotypes. Although AngII caused similar aortic dilations in C57BL/6 and MCP1
-/-
mice, deficiency of CCR2 attenuated aortic expansion to the same extent as previously described in hypercholesterolemic mice (P<0.001) .

Conclusion:
CCR2 reduces AngII-induced ascending aortic aneurysm formation in normolipidemic mice, although not via activation through MCP-1.
Abstract 405: Vascular Smooth Muscle Cells (VSMCs) from Abdominal Aortic Aneurysms (AAA) Avidly Take Up Oxidized Cholesterol (oxChol) with Unique Responses Compared with VSMCs from Nonaneurysmal Aorta (NAA)
Authors: Vemuri Chandu , Airhart Nathan , Bartlett Sarah , Wright Melissa , Arif Batool , Curci John A
Published: March 2022

Objective:
We sought to determine if VSMCs from AAA respond to oxChol exposure in a manner which may promote aortic matrix damage.

Methods:
Early passage VSMCs (5x10
5
) from human AAA, Carotid plaques (CP) and nAA were compared after exposure to media with oxChol for 72 hours. Cells were counted and stained with Oil Red O, and staining was graded on a 0-4 scale, by 5 blinded experts. Conditioned media (CM) was assayed by ELISA for MMP-2, and corrected for cell number (mean±SE).

Results:
The average grade of Oil Red O stain (Fig 1) of VSMCs from AAA was 2.89 +/- 0.225, CEA plaques was 3.14 +/- 0.151 and nAA was 1.76 +/- 0.258. Cholesterol uptake was significantly different for AAA vs. nAA (p=.015) and CEA vs. nAA (p=.0002), but not between the unstained controls. Cell number (x10
3
) at harvest after oxChol exposure was significantly greater than media alone for CP (562±48 vs 338±79, P<0.02) and nAA (343±49 vs 250±31), but there was no effect on AAA (339±49 vs 313±43). With exposure to OxChol, MMP-2 concentration in CM from AAA cells was similar to that from CP cells (224±51ng/ml vs. 352±46ng/ml), but production from nAA cells was significantly less than CP (178±61ng/ml, P<0.05) but not compared to AAA.

Conclusion:
VSMCs from human AAA exposed to oxChol had significantly increased cholesterol uptake, with MMP-2 production comparable to CP cells. Exposure to oxChol resulted in a mitogenic response in the CEA and nAA cells not seen in the AAA cells. Hypercholesterolemia may promote AAA due to pathologic cellular responses of increased cholesterol uptake, decreased growth and increased protease production. Additional studies are needed to clarify these responses.
Abstract 407: Role of Apoa1 in Regulating Mitochondrial Function in Acute Myocardial Infarction
Authors: Penn Marc S , Latchoumycandane Calivarathan , McIntyre Thomas M , Lesnefsky Edward J
Published: March 2022

Background:
High density lipoprotein (HDL) and apoA-I levels inversely correlate with risk of death from ischemic heart disease; however, the role of apoA-I in myocardial response to ischemia has not been well defined.

Methods and results:
To test whether apoA-I, the primary HDL apolipoprotein, has an acute anti-inflammatory role in ischemic heart disease, we induced myocardial infarctions in apoA-I
-/-
and apoA-1 heterozygotic animals. We observed a 52% increase in infarct size as a percent of area at risk in heterozygotic and a 125% increase in apoA-I null animals compared to wild-type mice. Mitochondrial oxidation contributes to tissue damage in ischemic reperfusion injury, and we found a significant defect in the electron transport chain of cardiac myocytes from apoA-I
-/-
mice. This was localized to the coenzyme Q pool that impaired electron transfer from complex II to complex III. Administration of CoQ10 to apoA-I null animals normalized the cardiac mitochondrial CoQ pool, and it reduced infarct size to that observed of wild type animals. CoQ10 administration did not significantly alter infarct size in wild-type mice.

Conclusions:
These data identify CoQ pool size and impaired mitochondrial function as major contributors to infarct size in the setting of low HDL/apoA-I. These data suggest a previously unappreciated mechanism for myocardial stunning, cardiac dysfunction, and muscle pain associated with low HDL/apoA-I levels that can be corrected by CoQ10 supplementation and suggest populations of patients that may particularly benefit from CoQ10 supplementation.
Abstract 409: Site-Specific Solvent Exposure of Major Apolipoproteins in Human High-Density Lipoprotein Complexes of Different Composition and Diameter
Authors: Gauthamadasa Kekulawalage , Greis Kenneth , Silva R A Gangani D
Published: March 2022

It is believed that the interactions of high density lipoproteins (HDL) with different plasma enzymes are partly determined by the site specific solvent exposure of apolipoproteins in HDL. Ultra-centrifugally isolated human HDL was separated into subpopulations containing both major apolipoproteins apoA-I and apoA-II (termed LpA-I/A-II) and only apoA-I (termed LpA-I) using thiopropyl sepharose chromatography. LpA-I/A-II and LpA-I were further separated into three sub-fractions based on the particle diameter utilizing fast performance liquid chromatography. Individual sub-fractions were subjected to partial acetylation; the extent of acetylation that represents the solvent exposure was quantified by mass spectrometry (MS). LpA-I of ∼85 Å, demonstrated solvent exposure variations for Lys residues in apoA-I in the range of 9-55%. Lys 133 (helix 5) and Lys 226 (helix 10) showed the highest solvent exposure of 54-55%. Surprisingly Lys residues 238 and 239 showed the lowest exposure of 9-10%. The rest of the Lys residues located in N-term through helix 10 ranged from 12-40% demonstrating distinctive differences among specific sites of exposure. The presence of apoA-II in comparable diameter LpA-I/A-II showed similar solvent exposure for apoA-I, implying the non-perturbing nature of apoA-II on apoA-I conformation. Limited proteolysis experiments resulted in similar fragment patterns for apoA-I in LpA-I and LpA-I/A-II, agreeing with mass spectrometry observations. Furthermore, proteolysis demonstrated that apoA-II in HDL is less susceptible to proteolysis compared to apoA-I in HDL. Confirming this observation further, the extent of acetylation of individual Lys residues of apoA-II in LpA-I/A-II demonstrated overall low solvent exposure (9-14%) by MS. Subtle solvent exposure differences were seen among particles of different diameter (75 Å
vs
110 [[Unable to Display Character: Ǻ]]). These novel findings bring out conformational details of major apolipoprotein in different HDL particles. Assessment of site specific solvent exposure of minor HDL apolipoproteins including apoE, apoD, apoCs, apoM and determination of their specific preference for LpA-I
vs
LpA-I/A-II are currently underway implementing high throughput mass spectrometry techniques.
Abstract 417: Changes in Endothelial Activation and Monocyte Mobilization and Recruitment in Collateral Growth Promotion/Impairment in Lean and Obese Mice
Authors: DiStasi Matthew R , Campana Gonzalo L , Ortiz Miguel A , Labarrere Carlos A , Miller Steven J , Ingram David A , Unthank Joseph L
Published: March 2022

Vascular compensation to arterial occlusion is compromised by cardiovascular risk factors including obesity and hyperglycemia. The specific mechanisms mediating this impairment remain unclear. We have previously observed hindlimb collateral growth (CG), the primary compensation to peripheral artery occlusion, to be profoundly suppressed in diet-induced obese (DIO) relative to lean mice (130 vs. 10% diameter enlargement) while anti-oxidant therapy (apocynin, Apo) restored CG capacity in DIO but suppressed it in lean mice. In this study we utilize lean and DIO mice, with and without Apo treatment to assess the hypothesis that CG is impaired in obesity via redox-dependent inhibition of endothelial cell activation and reduced mobilization and/or recruitment of requisite macrophages. The right femoral artery was ligated distally to induce the gracilis artery to function as the primary collateral. The gracilis muscles of the right (exptl) and left (control) limbs were harvested 3 days post-ligation and immunostaining was performed to evaluate endothelial cell activation (# of ICAM-1 positive microvessels/mm
2
) and macrophage recruitment (# of F4/80 positive cells/mm
2
). ICAM-1 immunoreactivity was increased in all experimental limbs but was significant only within the DIO and Lean+Apo groups which are characterized by suppressed CG. The number of F4/80 positive cells was increased in the gracilis muscle of the experimental limb of all groups with a greater increase in the DIO than in Lean and DIO+Apo. FACS analysis of circulating monocytes demonstrated an increase at 1-day post-ligation followed by a decrease at day 3, with a greater percent decrease occurring in the DIO compared to Lean. The data suggest that mechanisms unrelated to deficits in mobilization, activation or recruitment mediate CG impairment in DIO.
Abstract 418: Is Depression a Risk Factor for Peripheral Arterial Disease? Insights from the Heart and Soul Study
Authors: Grenon S Marlene , Hiramoto Jade , Vittinghoff Eric , Whooley Mary , Cohen Beth E
Published: March 2022

Objectives:
Risk factors for peripheral artery disease (PAD) are tightly linked to those for coronary artery disease (CAD). Although depression is known to increase risk of CAD, fewer studies have evaluated depression and PAD, and the mechanisms linking depression and PAD remain unknown. We examined the association of depression with prevalent PAD and with prospective PAD events and evaluated potential mediators of these associations.

Methods:
We used data from the Heart and Soul Study, a prospective cohort of 1,024 men and women with CAD recruited in 2000-2002 and followed for a mean of 7.2 years with <5% loss to follow-up. Depression was assessed with the validated nine-item Patient Health Questionnaire. Prevalent PAD at baseline was determined by self-report. Prospective PAD events were adjudicated based on physician diagnosis, radiological imaging and/or need for PAD surgery. We used logistic and Cox models to estimate the independent associations of depression with prevalent PAD at baseline and subsequent PAD events, adjusting for potential mediators of these associations.

Results:
At baseline, depression affected 19% of patients. Prevalent PAD was reported by 12.1% of patients with depression and 7.3% of those without (age-adjusted OR 1.79, 95% CI 1.06-3.03, p=0.03). In age-adjusted models, physical inactivity, diabetes, and CRP level each explained more than 5% of the association between depression and prevalent PAD. During follow-up, PAD events occurred in 9% of patients with depression and 6% of those without (age-adjusted HR 1.77, 95% CI 1.02-3.07, p=0.04). Factors explaining more than 5% of the association between depression and incident PAD events included inactivity, diabetes, prevalent PAD, race/ethnicity, smoking, HDL, and triglyceride levels.

Conclusions:
Depression was associated with a greater risk of PAD at baseline and with prospective PAD events, providing evidence that depression is an important risk factor for the development of PAD. Since elevations in traditional, modifiable cardiovascular risk factors partially explain these associations, such risk factors should be assessed and aggressively treated. Furthermore, this study emphasizes the importance of depression screening and treatment in patients with PAD.
Abstract 42: Monocyte Tissue Factor-Dependent Activation of Coagulation in Hypercholesterolemic Mice and Monkeys Is Inhibited by Simvastatin
Authors: Owens A Phillip , Passam Freda H , Antoniak Silvio , Marshall Stephanie M , McDaniel Allison L , Rudel Lawrence , Williams Julie C , Hubbard Brian K , Dutton Julie-Ann , Wang Jianguo , Tobias Peter S , Curtiss Linda K , Daugherty Alan , Kirchhofer Daniel , Luyendyk James P , Moriarty Patrick M , Nagarajan Shanmugam , Furie Barbara C , Furie Bruce , Johns Douglas G , Temel Ryan E , Mackman Nigel
Published: March 2022

Hyperlipidemia is associated with a prothrombotic state that includes activation of platelets. However, the molecular pathway that leads to the activation of the coagulation cascade has not been defined. Hyperlipidemia leads to the presence of low levels of oxidized lipoproteins, such as oxidized LDL (oxLDL), in the circulation and the accumulation of oxLDL in atherosclerotic lesions.. Monocytes and macrophages are activated by oxLDL binding to a TLR4/TLR6/CD36 receptor complex. We hypothesized that oxLDL induction of monocyte tissue factor (TF) expression and the release of TF-positive microparticles (MPs) produces a prothrombotic state. In support of this hypothesis, we found that oxLDL induced TF expression in human monocytic cells and monocytes. In addition, patients with familial hypercholesterolemia had elevated levels of plasma MP TF activity. Furthermore, a western diet induced a time-dependent increase in plasma MP TF activity and activation of coagulation in both LDL receptor-deficient mice and African green monkeys. Inhibition of TF or a genetic deficiency of TF in bone marrow cells reduced coagulation in hypercholesterolemic mice, consistent with a major role for monocyte TF in the activation of coagulation. Similarly, a deficiency of either TLR4 or TLR6 reduced levels of MP TF activity consistent with a role of the TLR4/TLR6/CD36 complex in oxLDL induction of monocyte TF expression. Importantly, simvastatin treatment of hyperlipidemic mice and monkeys reduced oxLDL, monocyte TF expression, MP TF activity, activation of coagulation, and inflammation without affecting total cholesterol levels. Our results suggest that the prothrombotic state associated with hyperlipidemia is caused by oxLDL-mediated induction of TF expression in monocytes via engagement of a TLR4/TLR6/CD36 receptor complex. Furthermore, simvastatin appears to act at multiple levels to reduce the prothrombotic state.
Abstract 420: Synthesis and Targeted Spatiotemporal Delivery of Peptide Amphiphile to the Vasculature
Authors: Kassam Hussein A , Moyer Tyson J , Chew Teng Leong , Stupp Sam I , Kibbe Melina R
Published: March 2022

Introduction:
The aim of this study is to develop and evaluate a novel systemically delivered targeted therapy that will prevent restenosis after all cardiovascular interventions. To accomplish this aim, our goal is to synthesize a self-assembled peptide amphiphile (PA) targeted to a unique collagen-binding sequence, and demonstrate binding to the site of vascular intervention. Our hypothesis is that our collagen binding PA will bind to the site of vascular injury.

Methods:
PA was synthesized using solid-phase peptide synthesis and purified by reversed-phase HPLC. The collagen-binding sequence and a fluorescent tag (AlexaFluor546) was incorporated into the PA. Vascular smooth muscle cells (VSMC) and adventitial fibroblasts (AF) were exposed to the PA (0.25-1.0 mM) in culture and metabolic activity was assessed with the MTT assay. Male Sprague Dawley rats underwent the carotid artery injury model followed by intra-arterial injection of the PA (5mg) for 10min. Carotid arteries and arterial cross-sections were imaged for fluorescence using excitation and emission wavelengths of 535 nm and 580 nm, resp.

Results:
VSMC and AF exposed to the targeted PA for 24hrs showed similar metabolic activity compared to controls except at the highest concentration (1mM) at which a 50% decrease was detected (P<0.05). Carotid arteries injured and exposed to intra-arterial PA followed by intraluminal flushing with PBS exhibited positive binding of the PA to the site of injury with 5-fold greater fluorescent intensity compared to background levels. Similarly, carotid arteries injured and exposed to PA in a similar manner followed by re-establishment of blood flow for 20min exhibited 3-fold greater fluorescent intensity than background levels. Arterial cross sections demonstrated binding of the PA to the intima. No significant fluorescent signal was detected in the uninjured arteries.

Conclusion:
These data demonstrate that our novel targeted PA is biocompatible with vascular cells in vitro. Furthermore, we successfully demonstrated specific binding of our injectable targeted PA to the site of injury. This research serves as the foundation upon which a targeted drug-eluting therapy will be designed and evaluated to prevent restenosis following vascular interventions.
Abstract 427: Transforming Growth Factor β/Smad3 Stimulates Smooth Muscle Cell Migration and Proliferation Through a Signaling Pathway that Involves C-X-C Chemokine Receptor Type 4 Expression and Activation of Mitogen-Activated Protein Kinase
Authors: Shi Daniel-Xudong , Seedial Stephen , Si Yi , Ren Jun , Sehgal Namratta , Suwanabol Pasithorn A , Liu Bo , Kent Craig K
Published: March 2022

Introduction:
Intimal hyperplasia is a complex process that requires vascular smooth muscle cell (SMC) migration and proliferation. We have previously shown that levels of transforming growth factor beta (TGF-β) and its signaling protein Smad3 are elevated following arterial injury and contribute to intimal hyperplasia. In this study we exam whether these effects might be mediated through a mechanism that involves the chemokine stromal cell derived factor (SDF-1α) and its receptor C-X-C receptor type 4 (CXCR4).

Methods and Results:
A microarray was used to better understand the signaling pathways through which TGF-β and Smad3 stimulate SMC proliferation and migration. Overexpression of Smad3 in SMCs followed by stimulation with TGF-β (5 ng/ml) for 6 hours resulted in significant alteration of ∼300 genes, the most dramatic effect being a >500 fold upregulation of CXCR4. Quantitative RT-PCR and Western blotting confirmed that Smad3/TGF- β increased CXCR4 transcription and protein expression by 155±9 and 24±5 -fold, respectively. Immunocytochemistry and flowcytometry analysis revealed a 6-fold increase in CXCR4 positive cells in TGF-β stimulated Smad3 overexpressing SMCs compared to control (P<0.05). TGF-β/Smad3 treated SMCs exhibited enhanced migration (4-fold VS control, P<0.01) in response to SDF-1α (100 ng/ml) which was eliminated after preincubation of SMCs with CXCR4 inhibitor AMD3100 (10 μM). Moreover, there was a 45±9% increase in proliferation (BrdU) in response to SDF-1α in TGF-β/Smad3 treated SMCs compared to GFP control (P<0.05). To explore the mechanism by which SDF-1α/CXCR4 produces proliferation and migration in SMCs, we studied the intracelluar signaling protein mitogen-activated protein kinase (MAPK), which is a known intermediate for both processes. Incubation of TGF-β/Smad3 SMCs with SDF-1α led to activation of MAPK (11-fold) as well as nuclear translocation of CXCR4 in a dose and time dependent manner.

Conclusions:
Our data demonstrate that TGF-β through Smad3 stimulates expression of CXCR4 in medial vascular SMCs. These SMCs in response to SDF-1α then proliferate and migrate to the subintimal layer. These findings suggest a novel pathway through which TGF-β enhances intimal hyperplasia at sites of arterial injury.
Abstract 430: Vascular Contrast Increases Permeability of Proximal Tubule Epithelium: A Possible Mechanism of Contrast-Induced Nephropathy
Authors: Yao Luyu , Kolluru Gopi K , Kevil Christopher G , Zhang Wayne W
Published: March 2022

Objectives:
Contrast Induced Nephropathy (CIN) is the third leading cause of hospital acquired renal failure and excludes numbers of patients from less invasive endovascular procedures. The mechanism of CIN is not fully understood. We investigated the hypotheses that CIN may result from direct toxicity or malfunction of kidney cells caused by contrast agent.

Methods:
Human kidney 2 (HK-2) cells were treated with Phosphate-Buffered Saline (PBS) or Iodixanol, a contrast agent, representing control and experimental groups. TUNEL assay was used to evaluate apoptosis. Cell proliferation was measured by BrdU incorporation. Expression of Claudin-2, a tight junction protein, was assessed by qRT-PCR and western blot. Intercellular gaps were measured using phase contrast microscopy and quantified by Image J software.

Results:
Iodixanol reduced tubule cell proliferation, but did not cause apoptosis. Phase contrast imaging of HK-2 cells demonstrated that intercellular gaps within the monolayer of cells were significantly increased by Iodixanol in a dose dependent manner. Western blot and qRT-PCR showed up-regulation of claudin-2 protein and mRNA expression. Both intercellular gap formation and up-regulation of claudin-2 expression are consistent with possible increase of permeability across proximal tubule epithelium, which is one of the known causes of renal failure.
Conclusion:
Our in vitro data do not support the hypothesis that direct kidney cell death from contrast agent is a major mechanism of CIN. However, increased permeability of proximal tubule epithelium caused by contrast may play an important role in CIN.
Abstract 433: Overexpression of Catalase Negates the Beneficial Effects of Dietary Lipids on Plasma Triglycerides and APOA1/C3/A5 Levels
Authors: Fei Jia , Wheaton Anne , Ennis Christopher , Cook Carla , Parthasarathy Sampath , Santanam Nalini
Published: March 2022

Linoleic acid (LA) and oxidized linoleic acid (OxLA) are major components of Western diet. Both these lipids exert their actions through induction of oxidative stress. Our earlier studies have shown that feeding these dietary lipids to C57Bl/6 mice for two weeks decreased plasma triglyceride levels and up-regulated ApoA5 levels. We hypothesized that decreasing oxidative stress by overexpressing antioxidant Catalase in mice will block these beneficial effects.
Methods:
24 male C57Bl/6 mice and 17 male Catalase (CAT) transgenic mice (8-12 weeks old) were divided into three groups each. Each group were fed orally either vehicle alone (ethanol), LA or OxLA (18mg/day) for 2 weeks. At the end of two weeks, the mice were sacrificed and plasma lipid profile along with liver mRNA and protein levels of Catalase and APOA1/C3/A5 was analyzed.
Results:
LA and OxLA feeding significantly decreased plasma triglycerides in C57Bl/6 mice but not other lipid cholesterols. However, no such changes were observed upon LA or OxLA feeding in CAT mice. The CAT mice expressed significantly higher mRNA levels of liver catalase but decreased APOA1/C3/A5 expression compared to C57Bl/6 mice, which was unaltered by LA or OxLA feeding. These effects were mediated by the modulation of the transcription factor, peroxisome proliferator-activated receptors (PPARs).
Conclusion:
In conclusion, overexpression of an antioxidant enzyme, catalase negated the beneficial effect of dietary lipids on triglyceride levels by altering the APOA1/C3/A5 pathway through modulation of PPARs. Our results suggest that a minimal level of oxidative stress is beneficial.
Abstract 447: Foam Cell Formation and Triglyceride Production After Incubation of Human Monocytes and Endothelial Cells with MAA-Modified Proteins
Authors: Garvin Robert P , Duryee Michael J , Hunter Carlos D , Shurmur Scott W , Klassen Lynell W , Thiele Geoffrey R , Anderson Daniel R
Published: March 2022

Introduction:
Oxidized proteins and the formation of foam cells are central aspects in the development and progression of atherosclerosis. Malondialdehyde acetaldehyde (MAA) modified low density lipoprotein (LDL) is generated when lipoproteins are exposed to the oxidative product malondialdehyde. These MAA-adducted proteins have been shown to bind scavenger receptors, up-regulate adhesion molecules, induce cytokine expression, and initiate antibody and T-cell responses in both human and animal models of atherosclerotic disease. However, the induction of foam cells using these antigens has been untested.

Objective:
The purpose of this study was to evaluate the ability of MAA-adducted proteins on LDL to induce foam cell formation and upregulate innate inflammatory mediators of atherosclerosis in human peripheral monocytes, mouse macrophage, and human aortic endothelial cell lines.

Methods:
Mouse aortic endothelial (2167), mouse macrophage (J774) and human peripheral monocytes were incubated with LDL, oxidized-LDL, MAA-LDL, or MAA-human albumin (25 and 50 ng/ml) for 24 or 48 hours and evaluated. Cells were accessed for uptake using Oil Red O, triglyceride production, and cholesterol esters using filipin. Real-time semi-quantitative RT-PCR and ELISA was performed to evaluate IL-6 and monocyte chemotactic protein-1 (MCP-1) mRNA expression and protein secretion. MAA binding was also assessed using an anti-MAA monoclonal antibody.

Results:
LDL uptake is increased in aortic endothelial cells, J774 and human monocytes as evidenced by filipin and Oil Red O staining for esterified cholesterol LDL as compared to sham controls. Triglyceride data show a 5.1 fold increase in monocytes and a 6 fold increase in aortic endothelial cells increase following MAA-LDL incubation over control p<0.001. Additionally, IHC for MAA-adducted proteins after incubation with MAA-modified demonstrate surface binding and intracellular uptake of MAA-adducted proteins. Incubation of MAA-albumin and MAA-LDL with aortic endothelial cells results in significant IL-6 and MCP-1 mRNA synthesis and protein expression 5 fold for IL-6 p<0.001 and 3 fold for MCP-1 p<0.01

Conclusions:
These data show that MAA-modified proteins bind to and are processed by endothelial cells, macrophage, and human monocytes. MAA-modified proteins result in the production of innate immune mediators of tissue inflammation and inflammation recruitment. The direct effect of MAA in forming “foam cells” as evidenced by increased triglyceride and cholesterol is supportive for a role of MAA-modified proteins in the progression of atherosclerosis. The nature and impact of MAA-modification is hypothesized to be central to cellular apoptosis, autophagy and/or necrosis.

Implications:
MAA-modified proteins may be directly pathogenic in the development of plaque inflammation and progression of atherosclerosis.
Abstract 450: Increased O-GlcNAc Modification Induces Vascular Calcification by Increasing Function of Runx2 and AKT
Authors: Heath Jack , Yuan Kaiyu , Sun Yong , Chen Jianfeng , Mao Xia , Chen Yabing
Published: March 2022

Objective—
Vascular calcification is prevalent in patients with atherosclerosis, diabetes mellitus, and end stage renal disease, and it increases morbidity and mortality of these patients. Dramatic vascular calcification has been demonstrated in several experimental diabetic models, including the streptozotocin-induced mouse model of diabetes. Using low-dose STZ, we demonstrated that STZ-induced hyperglycemia was associated with increased protein O-link glycosylation (O-GlcNAc) modification in the mouse vasculature. The present studies sought to determine the role of O-GlcNAc modifications in regulating vascular calcification.

Methods and Results—
We found that Thiamet-G, a specific inhibitor of O-GlcNAcase (OGA), the enzyme that removes O-GlcNAc, dramatically increased O-GlcNAc modification in primary mouse vascular smooth muscle cells (VSMC). Non-toxic concentrations of Thiamet-G (10nM - 10μM) induced calcification of VSMC in a dose-dependent manner. Thiamet-G-induced VSMC calcification was associated with increased expression of osteogenic genes, including the key regulator for VSMC calcification Runx2, osteocalcin, alkaline phosphatase, and collagen I (Runx2=2.97±0.31, OC=4.85±0.10, ALP=2.35±0.33, ColIa=1.86±0.20, p<0.05). In addition, Thiamet-G induced transactivity of Runx2 (fold=1.52±0.09, n=3, p<0.05) and O-GlcNAc modification of Runx2.

Furthermore, OGA shRNA markedly increased O-GlcNAc modification in VSMC. A 3.7-fold increase in calcification, as indicated by calcium content, was demonstrated in OGA knockdown VSMC compared with control VSMC. Increased calcification was associated with a 4.8-fold increase in expression of osteocalcin, a Runx2-regulated osteogenic gene. In addition, OGA knockdown enhanced and sustained phosphorylation/activation of AKT, an upstream signal that we have reported to regulate Runx2 activity.

Conclusion—
We have found that increased O-GlcNAc modification in VSMC enhances calcification of VSMC, which may be attributed to increased activation of AKT and Runx2 signals. These results provide novel mechanistic insights into the role of protein O-GlcNAc modification in regulating vascular calcification in diabetes.
Abstract 459: Attenuation of Experimental Atherosclerosis by Systemic Administration of Interleukin-19
Authors: Ellison Stephen P , Gabunia Khatuna , Kelemen Sheri E , Scalia Rosario , Autieri Michael V
Published: March 2022

Introduction:
IL-19 is a newly described Th2 interleukin which had previously been ascribed to be inflammatory cell-specific. Ours is the only laboratory to investigate a role for this interleukin in vascular biology. The hypothesis of this study is that IL-19 can attenuate atherosclerosis through the potential mechanism of dampening leukocyte-endothelial cell interactions.

Methods and Results:
IL-19 is not detected in normal artery, but is highly expressed in multiple cell types in atherosclerotic plaque suggesting a role for this interleukin in development of atherosclerosis. Five month old LDLR
-/-
mice were fed an atherogenic diet and injected i.p. with either 1.0ng/g/day IL-19, 10.0ng/g/day IL-19, or PBS for 12 weeks. Mice receiving systemic IL-19 injections developed significantly less atherosclerotic plaque in the aortic arch compared with control mice (17.7+/-1.7% vs 5.3+/-1.1%, p<0.0001 for 1.0ng/g/day, and 18.5+/-1.5% vs 2.2+/-0.3%, p<0.0001 for 10.0ng/g/day). To determine if IL-19 could cause plaque regression, 10.0ng/g/day IL-19 or PBS was administered to LDLR
-/-
mice which had previously been fed an atherogenic diet for 14 weeks. After 8 weeks of treatment, a significant difference of p<0.0025 between the no treatment and PBS groups, and a significant difference of p<0.0014 between PBS and IL-19 treated groups was noted, but no significant difference between the no treatment and IL-19 treated groups was observed. No difference in serum cholesterol, triglycerides, or body weight was noted between the control and IL-19 groups. Immunohistochemical staining of the aortic root from these mice for the macrophage marker F4/80 demonstrates IL-19 treated mice have significantly less macrophage infiltrate compared with mice injected with PBS (36.3+/-2.3% vs 20.0+/-2.7%, p<0.0004). Wild-type mice were fed an atherogenic diet and injected with 1.0ng/g/day IL-19 for 12 weeks, and intravital microscopy of leukocyte adhesion indicates that IL-19 decreases leukocyte-endothelial cell interactions in vivo.

Conclusion:
When taken together, these data suggest an important role for IL-19 in inhibition, but not regression, of atherosclerotic plaque in vivo. A potential mechanism is that IL-19 can decrease leukocyte-endothelial cell interaction.
Abstract 460: Thioredoxin-1 and Its Interaction Partners Control Apoptosis in the Endothelium
Authors: Eckers Anna , Zschauer Tim-Christian , Kunze Kerstin , Jakob Sascha , Altschmied Joachim , Haendeler Judith
Published: March 2022

In the endothelium, defense against oxidative stress as well as inhibition of apoptosis is mainly mediated by thioredoxin-1 (Trx-1). Trx-1 protein levels are reduced in aged endothelial cells (EC) in vitro and in vivo, leading to increased apoptosis sensitivity. Apoptosis is dependent on (i) cytoskeletal changes resulting in formation of stress fibers (thick bundles of actin), (ii) activation of caspase-3 and (iii) the redox status of the cell. The mechanisms how Trx-1 protects EC against apoptosis are not completely understood, but could involve protein-protein interactions.

We identified three proteins interacting with Trx-1: γ-actin, the p17 subunit of Caspase-3 and the AP-endonuclease 1/redox factor 1 (APEX1). We could show that H2O2-induced stress fiber formation in EC was prevented by exogenously applied or overexpressed Trx-1. Moreover, the interaction between Trx-1 and non-polymerized γ-actin precluded Trx-1 degradation, suggesting a mutual protection of both proteins. Secondly, we observed that Trx-1 overexpression reduces apoptosis induction by the p17 subunit of Caspase-3 in EC. This was corroborated by the identification of a disulfide bridge-dependent interaction of Trx-1 with cysteine 163 in p17 preventing association with the second subunit, p12, which is essential for full activation of Caspase-3. However, interaction of Trx-1 with other proteins is not necessarily dependent on disulfide bridge formation. We could demonstrate that APEX1 can protect EC against apoptosis. Interestingly, we found that Trx-1 and APEX1 associate with each other even after mutation of 3 (APEX1) or 2 (Trx-1) cysteines with the same efficiency as the wildtype proteins. It is conceivable that this interaction is required for the protective effect of APEX1.

Taken together, our data demonstrate the importance of Trx-1 in maintaining endothelial cell integrity mediated by interaction with other proteins, and that apoptosis protection of EC is not only due to its anti-oxidative capacity. Loss of Trx-1 in senescent EC therefore importantly contributes to the increased apoptosis sensitivity observed in during aging.
Abstract 470: Redox Rgulation of SIRTUIN1 in Endothelium-Dependent Vascular Function
Authors: Kim Cuk-Seong , Jung Saet-Byel , Hoffman Timothy A , Narqvi Asma , Irani Kaikobad
Published: March 2022

Objective:
The DNA repair enzyme/reducing protein Apurinic/Apyrmidinic Endonuclease 1/Redox Factor-1 (APE/Ref-1) maintains normal endothelial function, as mice heterozygous for APE1/Ref-1 (+/-) have impaired endothelium-dependent vasorelaxation and are hypertensive. APE1/Ref-1 maintains many redox sensitive transcription factors in their reduced active form. The SIRTUIN1 (SIRT1) protein deacetylase also promotes endothelial nitric oxide (NO) production and endothelium-dependent vasorelaxation. Although SIRT1 activity is subject to redox regulation, whether it is governed by APE1/Ref-1 is not known. The purpose of this study is to determine if APE1/Ref-1 governs the redox state and activity of SIRT1, and whether SIRT1 mediates the effect of APE1/Ref-1 on endothelium-dependent vascular function.

Methods and results:
In endothelial cells APE1/Ref-1 maintains cysteine (thiol) residues in SIRT1 in the reduced (free) form and is obligatory for endothelial SIRT1 activity. In vitro APE1/Ref-1 increases the free thiol content of SIRT1, targeting functionally critical cysteine residues in the catalytic domain of SIRT1 for reduction, and activates SIRT1. Cysteine residues in the N-terminal redox domain of APE1/Ref-1 are essential for activating SIRT1 and for increasing free thiol content of SIRT1. APE1/Ref-1 protects endothelial SIRT1 from hydrogen peroxide-induced oxidation and inactivation. Moreover, APE1/Ref-1 stimulates lysine deacetylation of nitric oxide synthase, a target of SIRT1 in endothelial cells. When compared to wild-type mice, APE1/Ref-1(+/-) mice have lower free thiol content of SIRT1, and diminished SIRT1 activity in tissues. Overexpression of SIRT1 in aortas of APE1/Ref-1 (+/-) mice restores endothelium-dependent vasorelaxation and vascular bioavailable NO to levels similar to those observed in wild-type mice.

Conclusions:
APE1/Ref-1 maintains endothelial SIRT1 in the reduced form, and is required for maintaining SIRT1 activity. This relationship between SIRT1 and APE1/Ref-1 is an important mechanism responsible for APE1/Ref-1-stimulated endothelium-dependent vasorelaxation.
Abstract 480: Activation of NRF2/ARE Pathway Regulates Hydrogen Peroxide Releasing NADPH Oxidase NOX4 in Endothelial Cells
Authors: Brunssen Coy , Eickholt Claudia , Langbein Heike , Brux Melanie , Goettsch Claudia , Goettsch Winfried , Arsov Alexander , Morawietz Henning
Published: March 2022

The NADPH oxidase isoform NOX4 produces mainly hydrogen peroxide (H
2
O
2
). The transcription factor NRF2 is a key mediator of cellular adaptation to redox stress. Regulation of NOX4 and formation of H
2
O
2
might be directly linked to NO release. NOX4 is the major endothelial NOX isoform and considered to be constitutively active. Regulation of NOX4 on transcriptional level by NRF2 might be directly linked to NO release and endothelial function.

Endothelial cells (HUVEC) were constantly exposed to high laminar shear stress (24h, 30dyn/cm
2
). This stimulates NO formation and leads to elongation of the cells in the direction of the flow. Previously, we could show that NOX4 is the major endothelial NOX isoform and downregulated by shear stress. Here we show that shear stress induces antioxidative response via upregulation of NRF2 which affects NOX4 expression. We transduced HUVEC with lentiviral particles containing short hairpin RNA (sh) against NRF2 and NOX4. Lentiviral downregulation of NOX4 using shNOX4 inhibited the shear stress-dependent elongation of cell shape in response to flow. Application of shear stress caused downregulation of NOX4 and upregulation of NRF2 and its target genes NQO-1 and HO-1. Attenuation of NRF2 by shNRF2 inhibited shear stress-dependent induction of NRF2 and its target genes. In addition, shNRF2 enhanced the shear stress-dependent downregulation of NOX4. Finally, we could show that downregulation of NOX4 is involved in the upregulation of eNOS and expression of NRF2 in response to flow. Nox4 overexpression had no effect on these processes. In conclusion, our data suggest a link between NOX4, NRF2-mediated antioxidative response and endothelial function.
Abstract 486: Soluble Biglycan Induces the Osteogenic Response in Human Aortic Valve Interstitial Cells via TLR2 and Mediates the Pro-osteogenic Effect of OxLDL
Authors: Song Rui , Zeng Qingchun , Ao Lihua , Yu Jessica A , Cleveland Joseph C , Fullerton David A , Meng Xianzhong
Published: March 2022

Calcific aortic stenosis affects a large number of people 65 or older. Our previous studies found that stimulation of TLR2 or TLR4 in human aortic valve interstitial cells (AVICs) up-regulates the expression of osteogenic mediators. Identification of endogenous agents that activate TLR2 and TLR4 in AVICs could lead to pharmacological intervention of calcific aortic stenosis. While biglycan and oxLDL accumulate in diseased areas of aortic valves, their role in the development and progression of this valve disease is poorly understood. Soluble biglycan is recognized as an endogenous ligand for TLR2 and TLR4 in macrophages. We recently found that oxLDL induces bone morphogenetic protein-2 (BMP-2) expression in human coronary artery endothelial cells. This study tested the
hypothesis
that soluble biglycan induces the osteogenic response in human AVICs via TLR2/4 and mediates the pro-osteogenic effect of oxLDL.
Methods and results:
The expression and release of biglycan were greater in AVICs of stenotic valves. Stimulation of cells with biglycan (0.05-0.20 μg/ml) increased the expression of BMP-2 and alkaline phosphatase (ALP). Further, prolonged stimulation with biglycan caused accumulation of calcium deposits in AVIC culture. Silencing of TLR2, not TLR4, markedly reduced protein levels of BMP-2 and ALP following stimulation with biglycan. Co-immunoprecipitation revealed the interaction of biglycan with TLR2. Biglycan induced the phosphorylation of ERK1/2 and p38 MAPK, and inhibition of either ERK1/2 or p38 MAPK reduced biglycan-induced expression of BMP-2 and ALP. Stimulation of AVICs with oxLDL (20 and 40 μg/ml) up-regulated biglycan expression and release. Neutralization or silencing of biglycan reduced the effect of oxLDL on the expression of BMP-2 and ALP.
Conclusions:
Extracellular soluble biglycan induces the osteogenic response in AVICs via TLR2. The ERK1/2 and p38 MAPK pathways are involved in mediating the pro-osteogenic effect of soluble biglycan. OxLDL stimulates the synthesis and release of biglycan by AVICs, and biglycan contributes to the mechanism underlying the pro-osteogenic effect of oxLDL on AVICs. These findings provide evidence supporting a pathobiological role of soluble biglycan and oxLDL in calcific aortic stenosis.
Abstract 498: Decreased High-Density Lipoprotein Cholesterol Levels and Macrophage Cholesterol Efflux Capacity in Mice Lacking Proprotein Convertase Subtilisin/Kexin Type 9
Authors: Choi Seungbum , Srivastava Ujala , Aljakna Aleksandra , Deng Bin , Peterson Blake R , Korstanje Ron
Published: March 2022

Background:
Proprotein convertase subtilisin/kexin type 9 (PCSK9) regulates low-density lipoprotein cholesterol catabolism and has been proposed as a promising therapeutic target for lowering low-density lipoprotein cholesterol levels in hypercholesterolemia patients. Several studies have shown that PCSK9 influences HDL cholesterol levels, however the molecular mechanism and potential consequence of targeting PCSK9 needs further study.

Methods and Results:
We isolated serum from Pcsk9 knockout (KO) mice to determine HDL cholesterol levels, HDL subunits and macrophage cholesterol efflux capacity. HDL cholesterol levels in Pcsk9 KO mice were significantly decreased in both sexes fed a normal chow and an atherogenic diet. HDL subunits were separated by gradient gel electrophoresis and HDL proteins were identified by mass spectrometry. Apolipoprotein E (APOE) was found in large HDL subunits in C57BL6/J (B6) mice, while it was absent in Pcsk9 KO mice. We found that Pcsk9 KO mice have decreased levels of serum APOE and increased LDLR in livers. We demonstrated that LDLR regulates serum APOE level by measuring this in mice containing gain- and loss-of-function in LDLR. APOE levels were decreased in Pcsk9 KO and human LDLR overexpressing transgenic mice, and increased in mouse containing non-functional LDLR. We tested the hypothesis that absence of APOE in HDL of Pcsk9 KO mice decreases macrophage cholesterol efflux capacity. Decreased fluorescently-labeled cholesterol efflux of Pcsk9 KO serum was demonstrated using both human THP-1 and mouse J774A.1 macrophage foam cells in vitro. We isolated hearts from Pcsk9 KO mice fed an atherogenic diet for 10 weeks until 34 weeks of age and determined atherosclerosis susceptibility by measuring atherosclerotic lesion size. The average volume of atherosclerotic lesions in the first 120 um of the ascending aorta was not significantly different between B6 and Pcsk9 KO mice.

Conclusion:
PCSK9 plays a critical role in regulating not only LDL, but also HDL in mice. The results demonstrate that increased LDLR level in Pcsk9 KO mouse liver likely decreases APOE in large HDL, which impairs macrophage cholesterol efflux capacity of Pcsk9 KO mouse serum.
Abstract 502: CSL112, a Novel Formulation of Human ApoA-I, Enhances the Capacity of Serum to Efflux Cholesterol from Macrophages
Authors: Gille Andreas , Diditchenko Svetlana , Leary Elizabeth T , Shear Chuck L , Rader Daniel J , Wright Samuel D
Published: March 2022

The ability of HDL to remove cholesterol from macrophages in atherosclerotic plaque is thought to underlie its inverse correlation with cardiovascular risk. Indeed, cholesterol efflux from macrophages is a functional serum marker that has an even stronger inverse association with coronary heart disease than HDL cholesterol (Khera et al. 2011, NEJM 364;2;127). We recently described CSL112, a novel formulation of full length human apoA-I and phospholipids designed to resemble nascent HDL and to maximize cholesterol efflux from macrophages. Here we studied the effects of CSL112 vs HDL3 on ex vivo cholesterol efflux in human plasma and serum. Addition of HDL3 to pooled plasma significantly increased ABCA1-independent efflux but had little or no effect on ABCA1-dependent efflux. In contrast, the addition of CSL112 increased not only ABCA1-independent efflux but also strongly increased ABCA1-dependent efflux in a concentration dependent fashion. Through a range of concentrations, CSL112 was significantly more efficacious in global cholesterol efflux than was HDL3. For example, at 5 μg/mL, CSL112 increased global efflux by 152% ± 5.6% vs 66% ± 7.4% for HDL3. Additional studies examined serum samples from five subjects with a range of lipid phenotypes including low, normal and high levels of HDL. When spiked into these sera, CSL112 increased both ABCA1-dependent and independent efflux in all subjects, regardless of lipid profile. In all subjects, the relative increase in ABCA1-dependent efflux was larger than that observed with ABCA1-independent efflux indicating selectivity of the interaction of CSL112 with the ABCA1 transporter. We speculate that the disproportionate rise in ABCA1-dependent efflux with CSL112 may be explained by the observation that PreBeta1-HDL levels measured by ELISA increase dramatically upon incubation of CSL112 with human plasma. We conclude that CSL112 robustly enhances the capacity of human serum to promote cholesterol efflux; the enhancement appears independent of lipid phenotype, is greater than that observed with HDL3 and that the efflux derives disproportionately from the ABCA1 pathway.