bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 227: Lipophagy’s About Face as Early Macrophage Foam Cells Transition to Late Stages
Authors: Netherland-Van Dyke Courtney , Romer Carrie , Jerome Gray (Jay)
Published: March 2022

Efflux of cellular sterol from macrophage foam cells plays an important role in limiting atherosclerotic lesion progression. Recently, lysosomes were shown to participate in efflux of cytoplasmic lipid droplet (LD) sterol through lipophagy. Lipophagy is a form of autophagy that mobilizes cholesteryl ester (CE) and triglyceride (TG) from LDs to lysosomes for degradation. In early atherosclerosis, lipophagy helps clear cytoplasmic sterol. However, in advanced atherosclerosis, macrophage lysosomes are impaired by excess sterol. We hypothesized that lipophagic delivery of LD CE to impaired lysosomes adds to lysosomal sterol burden exacerbating lysosome dysfunction and cellular sterol accumulation rather than promoting sterol clearance. To test this, we induced lysosome CE loading in THP-1 macrophages with aggregated LDL (aggLDL). This elevated lysosomal pH and led to accumulation of autophagic flux markers (LC3 and p62, >2 fold) and the LD coat protein adipophilin (6 fold) in lysosomes without altering lysosomal and upstream autophagic markers. This suggests lipophagy remained active despite the lysosome dysfunction and contributed to lysosomal sterol buildup. Triglyceride rich particles (TRP) have been shown to restore lysosome function and promote clearance of excess sterol from lysosomes and cells. If lipophagy is quantitatively important in late stage foam cells, TRP should reduce the aggLDL-induced lysosomal accrual of lipophagic markers. VLDL treatment of aggLDL-loaded cells restored lysosomal pH and cleared cellular sterol, in addition to, decreasing LC3-II and p62 in whole cells and isolated lysosomes (>2 fold) and reducing the fluorescent colocalization of LC3 with lysosomes by 37.9%. VLDL also increased autophagic LD targeting (26.3% LC3 colocalization) and delivery to lysosomes (24.6% LAMP-1 colocalization) for degradation. In conclusion, TRP restores lysosome function including the hydrolysis and clearance of LD CE through lipophagy in aggLDL-loaded macrophages. These data indicate that lipophagy is a potential target for therapy in late-stage foam cells but only as an adjunct to interventions that restore lysosome function.
Abstract 237: CAT-2003 is a Novel Small Molecule That Activates Lipoprotein Lipase and Reduces Fasting and Postprandial Triglycerides
Authors: Liu Feng , Picarella Dominic , Zimmer Mike , Bista Pradeep , Lee Diana , Vu Chi , Jirousek Michael
Published: March 2022

Introduction:
Triglycerides from chylomicron and VLDL particles are cleared in the periphery through Lipoprotein lipase (LPL). LPL is negatively regulated by Angiopoietin-like proteins 3 and 4 (Angptl3/4) and Apolipoprotein C3 (apoC3). CAT-2003 is an orally active small molecule that reduces fasting and postprandial triglycerides (TGs) in rat models of hypertriglyceridemia. We hypothesize that CAT-2003 activates LPL by reduction of Angptl3/4 and apoC3.

Methods and Results:
In vitro addition of CAT-2003 to THP-1 cells increased heparin releasable LPL activity rapidly (28% increase at 2 hr, p=0.01) and was sustained (52% increase at 20 hr, p=.0002). Angptl4 mRNA was decreased, while levels of LPL mRNA remained unchanged. In HepG2 cells CAT-2003 lowers apoC3 (94%, p<0.001) and Angptl3 (99%, p<0.001) mRNA. Secretion of newly made apoC3 protein was significantly inhibited (54%, p=0.004) within 1 hour of CAT-2003 treatment and was reduced by 84% (p<0.0001) after 4 hours. In vivo administration of a single oral dose of CAT-2003 (100 mg/kg) reduced plasma TGs excursion by 80% (p<0.014) at 2 hours and by 91% (p<0.031) at 4 hours following an oral oleate challenge in Sprague-Dawley (SD) rats (n=4 per arm). In a separate SD rat study CAT-2003 (30 mg/kg) was administered as a single oral dose and a time course (0.5 to 4 hrs, n=4 per time point) of PCSK9, apoC3 and Angtpl4 gene regulation in intestine and liver was studied. Expression of these genes was reduced as rapidly as 30 minutes and the reduction was sustained through 4 hours. In a 5-day study in Zucker fa/fa rats with TGs elevated above 900 mg/dL, qd oral dosing with CAT-2003 (10, 30, 100 and 300 mg/kg, n=8 per arm) resulted in dose-dependent reductions in fed and fasted plasma TGs with fasting reductions ranging from 41% (p<0.01) at 10 mg/kg to 67% (p<0.0001) at 300 mg/kg.

Conclusion:
CAT-2003 inhibits the negative regulators of LPL in vitro and in vivo. The time course of action is rapid and sustained in vitro with a translation to in vivo SD and fa/fa rats. CAT-2003 offers a potential new therapeutic for the treatment of severe hypertriglyceridemia and is being evaluated in Phase 2 clinical studies.
Abstract 247: Dipeptidyl Peptidase-4 Inhibition With Sitagliptin Acutely Inhibits Intestinal Lipoprotein Particle Secretion in Healthy Humans
Authors: Xiao Changting , Dash Satya , Morgantini Cecilia , Patterson Bruce W , Lewis Gary F
Published: March 2022

Postprandial dyslipidemia, in part due to overproduction of triglyceride-rich lipoprotein (TRL) particles from the liver and the intestine, contributes to increased CVD risk. The DPP-4 inhibitor sitagliptin, an anti-diabetic agent, has been shown to reduce postprandial lipid excursion following a mixed meal. The underlying mechanism of this effect, however, has not been examined in humans. This study was designed to examine intestinal and hepatic TRL particle production and clearance in response to a single oral dose of sitagliptin. 15 lean, healthy male volunteers were studied in two occasions, 4-6 weeks apart, receiving sitagliptin (100 mg) or placebo in random order. Kinetics of TRL particles of intestinal and hepatic origin were measured using stable isotope tracer infusion techniques and with control of pancreatic hormone levels. Sitagliptin decreased TRL apoB-48 concentration (-32%, P<0.05) by reducing production rate (sitagliptin 47.2+/- 11.0 vs placebo 93.9 +/- 25.2 ug/kg/d, P<0.05). Fractional catabolic rate was not significantly affected. TRL apoB-100 concentration, production rate and fractional catabolic rate were not significantly different between sitagliptin and placebo. Plasma TG, free fatty acids, glucose, pancreatic hormones, and TRL TG were similar between treatments. In conclusion, sitagliptin acutely inhibits intestinal, but not hepatic, lipoprotein particle production, independent of changes in pancreatic hormones and circulating glucose and free fatty acids. This pleotropic effect of sitagliptin explains in part the reduction in postprandial lipemia seen in clinical trials and may provide metabolic benefits beyond glucose lowering.
Abstract 259: Inflammation and Preoxidation Are Increased in High-Density Lipoproteins From Chronic Heart Failure Subjects
Authors: Henry Sarah-Louise , Fogarty Mark , Ko Angela , Ingle Lee , Cleland John G , McEneny Jane
Published: March 2022

Chronic heart failure (CHF) is a global term for the physiological state in which cardiac output is insufficient to meet the needs of the body and lungs. While oxidative stress, defined as an excess production of reactive oxygen species relative to antioxidant defence, has been shown to play an important role in the pathophysiology of CHF. However, although high density lipoproteins (HDL) have important cardioprotective properties, inflammation and oxidative stress can induce dramatic changes, which negate their antiatherogenic properties. Thus, such impairment to HDL may provide an important link between inflammation and oxidative stress in the pathogenesis of CHF.

This pilot study investigated the antiatherogenic properties of HDL in CHF.

Ten control subjects (BNP<100 nM) were matched for age and gender with 22 CHF subjects (BNP>100 nM). HDL
2
and HDL
3
were isolated from serum by rapid ultracentrifugation. SAA was measured by an ELISA procedure, paraoxonase-1 (PON-1) activity, hydroperoxide (HPO) and conjugated diene (CD) concentration were measured by spectrophotometric assays.

Results:
Compared to the control subjects, BNP was higher in the CHF subjects [50 (11) vs. 2439 (900) nM; p=0.015] and in HDL
2
and HDL
3
, SAA-related inflammation was increased, while the activity of the antioxidant enzyme PON-1 was decreased and CDs, one of the markers of lipid peroxidation, were increased (see table).

Conclusion:
This pilot study has shown that HDL displayed a proatherogenic phenotype in CHF subjects, which we suggest may be biologically relevant in the pathophysiology of CHF.
Abstract 260: Interruption of Protein-Protein Interaction of CD36 With Other Proteins Alters CD36 Biological Functions
Authors: Huang Wenxin , Li Renhao , Ramakrishnan Devi Prasadh , Silverstein Roy L
Published: March 2022

CD36 is a transmembrane glycoprotein belonging to the type 2 scavenger receptor family. On macrophages and platelets CD36 recognizes oxidized LDL and other endogenous danger signals and thereby transmits signals that promote athero-thrombotic processes. CD36 has been shown to associate physically with other transmembrane proteins, including integrins, tetraspanins, and toll-like receptors, and CD36-mediated cell signaling is modulated by these partner molecules. The CD36 N-terminal transmembrane domain (nTMD) contains a GXXXG sequence motif that in several membrane proteins has been shown to mediate protein-protein interactions. We thus hypothesized that the nTMD is involved in CD36 protein-protein interactions. We detected CD36 interactions with specific macrophage surface proteins with an immunofluorescence-based cross linking assay (Proximity Ligation Assay®/PLA) and confirmed them by immunoprecipitation/immunoblot (IP/IB). Prior to these assays cells were incubated with a synthetic 29 amino acid peptide containing the 22 amino acid of CD36 nTMD or a control peptide in which the glycine residues in GXXXG motif were replaced by valine. In functional experiments, macrophages were pre-incubated with peptides and then treated with oxLDL or native LDL prior to assessing LDL uptake at 1hr, foam cell formation after 24hr by oil red O staining, ROS generation by fluorescence probes, and cell migration in a modified Boyden Chamber Assay. CD36 nTMD peptide treated cells compared to untreated or control peptide treated cells had decreased CD36 surface associations with both CD9 and CD18 as detected by both PLA and IP/IB. Functional assays revealed that CD36 nTMD peptide significantly decreased DiI-labeled oxLDL uptake and decreased foam cell formation by 44% (p <0.0001). This was associated with a 32% decrease in oxLDL-mediated ROS generation (p < 0.001). OxLDL-induced inhibition of macrophage migration was inhibited by 84% (p < 0.0001) by TMD treatment compared to the control peptide. In summary, these data support the hypothesis that CD36 nTMD mediates interaction of CD36 with other proteins such as CD9 and CD18, and that altering these interactions in macrophages ameliorates pathologically important CD36 mediated responses to oxLDL.
Abstract 261: Inhibitory Effect of Isosorbide Mononitrate on the Formation of Atherosclerosis in Low-Density Lipoprotein Receptor and Heme Oxygenase-2 Double Knockout Mice
Authors: Ishikawa Kazunobu
Published: March 2022

Objective:
Isosorbide mononitrate (ISMN), a well-known nitrate, have been widely used in the management of ischemic heart disease. Although it significantly alleviate angina pain, development of tolerance and its pro-oxidant properties have been provoking continuing debate. To this day, the effect of nitrate therapy on atherosclerotic formation remains unsolved. To elucidate this clinically-important problem, we examined the effect of ISMN treatment on the atherosclerotic formation using high-fat fed low density lipoprotein (LDL) receptor and heme oxygenase-2 double-deficient (LDL-R/HO-2 -/-) mice, in which only anti-oxidative and anti-inflammatory isoform of heme oxygenase-1 (HO-1) had been expressed in atherosclerotic arterial wall cells. We also examined the role of bilirubin, a product of HO-1 reaction, in these ISMN models.

Methods:
During 6 week high-fat diet, LDL-R/HO-2 -/- mice were treated with ISMN (2 mg/kg/day or 200 mg/kg/day). Control animals were treated with vehicle. After sacrifice, resultant atherosclerotic formation was determined. We also examined the expressions of anti-oxidative enzyme such as HO-1 and extracellular superoxide dismutase (ecSOD), changes of reactive oxidative metabolites (d-ROM) in plasma. We also examined the effects of ISMN on bilirubin formation using cultured endothelial cells.

Results:
ISMN treatments did not alter plasma lipid profiles. ISMN treatment (200 mg/kg/day) significantly inhibited atherosclerotic lesion formation with marked HO-1 induction in aortic wall. In contrast, we did not observe the changes of extracellular superoxide dismutase (ecSOD) expression. ISMN treatments did not increase the levels of plasma d-ROM. However, ISMN treatments significantly increased bilirubin production in cultured endothelial cells.

Conclusions:
These results suggest that optimal ISMN treatment significantly inhibit atherosclerotic lesion formation via HO-1 induction although the role of bilirubin in these models needs to be further elucidated.
Abstract 264: High Serum Lp(a) Levels Are Associated With Subclinical 3-Vessel and Left Main Coronary Disease, Severe Stenoses and Plaque Volume in Apparently Healthy African Americans
Authors: Kral Brian G , Becker Lewis C , Qayyum Rehan , Vaidya Dhananjay , Yanek Lisa R , Fishman Elliot K , Becker Diane M
Published: March 2022

Background:
Serum Lp(a) is a CAD risk factor in European Americans (EA). Levels are 2-3 fold higher in African Americans (AA) but associations with CAD are inconsistent, including coronary calcium. The relationship of race-specific levels of Lp(a) with the extent and severity of subclinical total coronary plaque (TCP) has not been described.

Methods:
We screened 418 apparently healthy individuals for risk factors and coronary plaque using advanced CTA. TCP volumes (mm3) were quantified using a validated automated method. Lp(a) was measured by ELISA. The association of Lp(a) with plaque was investigated by race. Multivariable modeling was performed with adjustment for traditional CAD risk factors, including LDL-C, and intrafamilial correlations.

Results:
Mean age was 51±11 years; 57% female; 34% AA. Lp(a) was higher in AA than in EA (median 38 [18,65] vs 17 [10,33], p<0.0001). Plaque was present in 45% of AA and 47% of EA. In those with plaque continuous Lp(a) levels were associated with plaque volume in AA (β=8.83, p<0.0001) but not EA (β=1.49, p=0.45). Lp(a) was strongly associated with plaque extent and severity (Table), and with TCP volume in AA (adjusted p=0.01) but not in EA (p=0.10). In AA, Lp(a)≥60 mg/dL (top quintile) was independently associated with a 3.7 times greater risk of 3-vessel and/or LM disease (95% CI:1.4-10.3), a 6.6 times greater risk of having a stenosis ≥50% (95% CI:1.4-38.4, p=0.02), and higher TCP (median 438 [203,1247] vs 142 [73,262], p=0.001).

Conclusion:
High Lp(a) is strongly associated with coronary plaque extent, severity, and volume in apparently healthy AA. High levels of Lp(a) may be particularly important in the pathogenesis of CAD in AA.
Abstract 275: Hypoxic Conditions Regulate the Expression and Activity of Coagulation Factors Important in Atherothrombosis
Authors: Stavik Benedicte , Halvorsen Bente , Iversen Nina , Dahl Tuva B , Sandset Per Morten , Skretting Grethe
Published: March 2022

Neovascularization is an important feature in advanced atherosclerosis. It is recognized by the formation of new blood vessels recruited from the adventitia that penetrate the plaque, presumably due to lack of oxygen (hypoxia) in the plaque core. These immature, leaky neovessels can cause intraplaque hemorrhages, and promote plaque vulnerability and rupture. Plaque rupture leads to activation of coagulation and the formation of a thrombus, which is the main contributor to acute manifestations, morbidity, and mortality in atherosclerotic disease. The extrinsic coagulation activator tissue factor (TF) and its inhibitor tissue factor pathway inhibitor (TFPI) have been detected in atherosclerotic lesions, and are expressed by macrophages, smooth muscle cells, and endothelial cells overlaying the plaques. We aimed to investigate how hypoxic conditions affected the endothelial expression and activity of coagulation factors important in the initiation of coagulation. Hypoxia was induced in primary human umbilical (HUVEC) and aortic (HAEC) endothelial cells with chemicals (DMOG/CoCl2) or 1% oxygen (O2) concentration, and mRNA and protein expressions were measured using qRT-PCR and ELISA, respectively. Microscopy of fluorescence labeled cells was used to visualize cell-associated TFPI. Cell-surface FXa activity was measured using a two-stage chromogenic substrate method. Results showed that hypoxia reduced the TFPI mRNA and protein levels in a dose-dependent manner. The effect was apparent on all the protein pools of TFPI; the secreted, the cell-surface associated, and the intracellularly stored, indicating that the regulation occurred at the transcriptional level. The TF mRNA expression was also affected in a dose-dependent way, where low doses of DMOG and CoCl2 resulted in an increased expression, while high dose of DMOG and 1% O2 reduced the TF mRNA levels. Activated endothelial cells showed a dose-dependent regulation of TF expression following hypoxic stimulation, which is identical to the regulation of the FXa activity in the cells. Our findings indicate that hypoxic conditions can influence the coagulant activity of endothelial cells, which may potentially promote clinical events and thus the severity of atherosclerotic disorders.
Abstract 289: Sex Hormones Influence Progression of Angiotensin II-Induced Abdominal Aortic Aneurysms in Hypercholesterolemic Mice
Authors: Cassis Lisa A , Wang Yu , Al-Siraj Yasir , Daugherty Alan , Thatcher Sean E
Published: March 2022

Objectives:
Similar to humans, angiotensin II (AngII)-induced abdominal aortic aneurysms (AAA) exhibit marked sexual dimorphism with a greater prevalence in male than female mice. We demonstrated that castration of male mice reduced formation of AngII-induced AAAs, while castration of females had no effect. However, the role of sex hormones (testosterone or estrogen) on the progression of an established AAA is undefined. We tested the hypothesis that sex hormones influence the progression of AngII-induced AAAs.

Methods/Results:
Male ApoE-/- male mice were infused with AngII (1,000 ng/kg/min) for 28 days to induce AAAs, stratified to sham-operated or castration groups, and subsequently infused with AngII for 56 days. Aortic lumen diameters were decreased significantly in castrated compared to sham controls (1.9 ± 0.1 vs 1.6 ± 0.1 mm, P<0.05). While external AAA diameters were not different between groups, 3D image analysis revealed increased wall/lumen ratios of AAAs from castrated compared to sham controls (9.5 ± 2.0 vs 4.8 ± 0.9 ratio; P<0.05). Increased AAA wall thickness was associated with more pronounced α-actin immunostaining and increased matrix deposition. In contrast, castration had no effect on AngII-induced atherosclerosis. LDLr-/- female mice that received a single dose of testosterone at 1 day of age to promote adult AAA susceptibility were infused with AngII. Females with an established AAA were stratified to sham-operated, castrated, and castrated+estrogen (E
2
, 36 μg/ml) groups for 56 days of AngII infusions. Castration of females significantly increased aortic lumen diameters compared to castrated females administered E
2
(vehicle, 2.2 ±0.2; E
2
, 1.7 ± 0.1 mm, P<0.05). Moreover, E
2
administration significantly decreased external aortic diameters (castrated, 2.6 ± 0.3; castrated+E
2
, 1.7 ± 0.1 mm, P<0.05). Finally, castrated females administered E
2
had significantly decreased atherosclerosis compared to sham controls (38.9 ± 3.6 vs 49.6 ± 2.3 % lesion surface area; P<0.05).

Conclusions:
These results indicate that testosterone contributes to AAA progression in males whereas E
2
administration lowered AAA progression in female mice.
Abstract 291: Upregulation of Multiple DNA Methyltransferase Isoforms is Associated With Tissue Inflammation in Human Thoracic Aortic Aneurysm
Authors: Hagler Michael A , Jbeli Aiham H , Kunkala Meghana , Roos Carolyn M , Sundt Thoralf M , Miller Jordan D
Published: March 2022

Thoracic aortic aneurysm (TAA) is a degenerative condition of the aorta characterized by aortic dilatation secondary to tissue inflammation and matrix remodeling, which ultimately progresses to aortic dissection and rupture. While recent work suggests that in vitro exposure to pro-inflammatory stimuli promotes DNMT3b-dependent DNA methylation in skeletal muscle, epigenetic mechanisms regulating gene expression in TAA remain largely uncharacterized. We therefore tested the hypothesis that expression of tumor necrosis factor α (TNF-α) is increased in human TAA tissue and that such increases are associated with upregulation of DNA methyltransferase (DNMT) levels. Normal aortic samples (n=13) were acquired from non-aneurysmal hearts/aorta not suitable for cardiac transplant, whereas TAA samples from patients with tricuspid aortic valves (n=29) were acquired from patients undergoing elective surgery for aortic aneurysm. We measured relative expression of TNF-α, DNMT1, DNMT3a and DNMT3b mRNA levels in normal and TAA tissue by qRT-PCR, and DNMT3b protein levels by western blot. Marked increases in expression of the inflammatory marker TNF-α were noted in TAA tissue (12.3± 2.34), and were associated with significantly increased DNMT1 (1.4±0.13), DNMT3a (1.44±0.09) and DNMT3b (1.94±0.27) mRNA levels (p<0.05 for all). DNMT3b protein levels were also dramatically increased in human TAA samples (2.99±0.58-fold, n = 10/group, p<0.05) when compared to normal aortic tissue. Collectively, these data suggest a novel potential role for DNA methyltransferases as major regulators of altered gene expression in TAA, and support our working hypothesis that inflammation-induced increases in DNMT3b levels may be critical repressors of protective gene expression in TAA.
Abstract 300: Mesenchymal Stem Cells Attenuate High-Mobility Group Box 1 to Inhibit Aortic Aneurysms
Authors: Sharma Ashish K , Lu Guanyi , Salmon Morgan , Murphy Michael , Ailawadi Gorav , Upchurch Gilbert R
Published: March 2022

Objective:
Mesenchymal stem cells (MSCs) have been shown to attenuate aortic aneurysms (AAs) in animal models, but their precise mechanism of action remains undefined. In this study, we hypothesize that MSCs modulate NADPH oxidase activity in macrophages to mitigate high mobility group box 1 (HMGB1; a proinflammatory nuclear protein) secretion causing attenuation of vascular inflammation and AA formation.

Methods:
Abdominal aortic tissue from male patients was obtained during open surgical repair and control aortic tissue was obtained from transplant donor patients. Human aortic tissue explants (or primary murine macrophages; 5x10
5
cells/well) were treated in vitro with elastase for 5 minutes, with or without co-culture with MSCs (1x10
5
cells per well) and supernatants were analyzed after 24hrs. In the murine in vivo model, AAs were induced in 8- to 12-week old C57BL/6 (WT) mice using an elastase-perfusion or heat inactivated elastase, with or without MSC treatment (given i.v. on day 1). Aortic tissue (harvested on day 14) or culture supernatants were analyzed for proinflammatory cytokine and HMGB1 production, as well as NADPH oxidase activity (measured by superoxide anion generation). Groups were compared using ANOVA followed by Bonferroni post hoc test.

Results:
Aortic tissue from human AA patients had a significant increase in HMGB1 levels compared to control (50.7±4.8 vs. 26.8±2.5 ng/ml, respectively; n=9-11per group; mean +/- S.E.). HMGB1 production and NADPH oxidase activity in the elastase-treated human aortic tissue explants were significantly attenuated by co-culture with MSCs (63.7± 5.3 vs. 19.6±6.3 ng/ml and 1673±48 vs. 317± 89 Relative Light Units; respectively, n=5). In the murine AA model, HMGB1 expression in the aortic tissue from the elastase-perfused WT mice was significantly attenuated by MSC treatment (79.6±3.6 vs. 33.4±3.6 ng/ml). As compared to elastase-treated macrophages, the co-cultures of macrophages with MSCs significantly attenuated cytokine (IL-23, TNF-α and HMGB1) production as well as NADPH oxidase activity (1140±73 vs. 268±25 RLU).

Conclusions:
HMGB1 production from macrophages plays an important role in AA formation and represents a novel target to protect against aneurysm formation by treatment with MSCs.
Abstract 305: Myeloid Cell-Specific VEGF-A Deletion Inhibits Experimental Abdominal Aortic Aneurysms
Authors: Xu Baohui , Fujiyama Naoki , Glover Keith , Xuan Haojun , Furusho Yuko , Tanaka Hiroki , Gerritsen Mary , Dalman Ronald
Published: March 2022

Mural macrophage infiltration and angiogenesis are histopathological hallmarks of abdominal aortic aneurysms (AAA). Macrophages produce angiogenic cytokine VEGF-A in many pathologic settings. It is not known whether VEGF-A produced by macrophages plays a role in aneurysm disease. In this study, we generated myeloid cell-specific VEGF-A conditional knockout (CKO) mice using the Cre/LoxP technology, and evaluated the influences of myeloid VEGF-A ablation on the formation and progression of aneurysm disease. AAAs were created in 10-12 week old male CKO and wild type (WT) mice via intra-aortic porcine pancreatic elastase (PPE) infusion and assessed by serial ultrasound measurements of aortic diameters followed by histology at sacrifice. Following PPE infusion, aneurysmal degeneration was significantly blunted in CKO mice. Histologically, medial elastin fragmentation, macrophage density, and mural angiogenesis were significantly diminished in the aortae of CKO mice compared to those in WT mice. Additionally, myeloid VEGF-A deletion caused marked reduction in peritoneal CD115-positive or F4/80-positive macrophages following thioglycollate injection. In conclusion, targeted deletion of VEGF-A in myeloid cells suppresses aneurysm initiation and progression in association with reduced mural macrophage accumulation and angiogenesis.
Abstract 307: Estimation of Femoral Arterial Diameters in Men and Women Using New Postprocessing MR Angiographic Fly-Through Technique
Authors: Kasam Mallikarjun
Published: March 2022

Background and Purpose:
Development of the human vasculature database including femoral arterial parameters like maximum femoral artery diameter (MFAD), length, branching angles, etc for both men and women will be critical for Peripheral Arterial Disease (PAD) diagnosis for a treating clinician. Sporadic or limited work has been reported in clinical/pre-clinical research till date. This work was an important effort towards the development of human vasculature database using our newly developed method for estimation of stenosis
1
.

Methods:
Patient exams were performed under an IRB-approved protocol. Raw MR angiographic images of 30 male & 30 female PAD patients were retrospectively processed and analyzed for MFAD. The measurement includes healthy as well as symptomatic regions of interest at different femoral arterial sections. MR angiographies with 1mm3 spatial resolution were used for this work. Clinical exams were acquired on 3.0 T scanners (GE, DVMR 20.1IB, Milwaukee, WI).

Data analysis:
The angiogram was post-processed using tree analysis and virtual endoscopy modules of Analyze 11.0 software (Analyze 11.0; Biomedical Imaging Resource, Mayo Clinic, Rochester, MN) to fly through the femoral artery. A statistical parameter called Brightness Area Product (BAP) was defined as the sum of the intensities above the sample minimum intensity/threshold set by the user. This BAP is treated as a metric for the assessment of degree of stenosis. Different parameters were tested using 2-way Anova student t-test.

Results and Discussions:
A statistical significance (p<0.05) has been noticed for MFAD and linear arterial measurements among men and women for both healthy and symptomatic sections of the peripheral artery. More work towards the development and validation of this study is under progress.

Reference:
1.

New Reconstruction Technique for MRA to "Fly Through" the Lumen of the Cerebral Vasculature Mallikarjunarao Kasam; Siva S Ketha; Soumya Konduru, ATVB 2013, FL, USA, Abstract 273.
Abstract 308: Ischemic Stroke and the Weekend Effect: A Population-Based Study
Authors: Khoshchehreh Mahdi , Malik Shaista
Published: March 2022

Objective:
We assessed in-hospital mortality and utilization of invasive procedures following ischemic stork admissions on the weekend versus weekdays in the United States.

Background:
stroke is a common condition requiring urgent diagnosis and intervention. Previous smaller studies show poor outcomes for time-sensitive medical conditions during weekend admissions, however whether these associations persist is not known.

Methods:
We used the 2009 Nationwide Inpatient Sample to examine differences in all-cause in-hospital mortality between weekend versus weekday admitted patients with a principal diagnosis of ischemic stroke. Hierarchical analysis was used to identify if weekend admission was associated with decreased utilization of procedural intervention and increased subsequent complications as well.

Results:
We identified 561,960stroke admissions of which 20.63% were weekend admissions. Adjusted mortality for the weekend admitted cohort was higher for weekend admissions [Odds Ratio (OR), 1.26; 95% Confidence Interval (CI) 1.17-1.37] and patients were significantly less likely to receive Endarterectomy or Thrombolytic therapy on their first day of admission [OR: 0.62, 95% CI: 0.60-0.64]. Further, for stoke patients admitted during the weekend who underwent procedural interventions, in-hospital mortality and complications were higher as compared to patients undergoing the same procedures on weekdays.

Conclusion:
For stroke patients, weekend admission is associated with higher mortality and lower utilization of invasive procedures, and those who did undergo these interventions had higher rates of mortality and complications than their weekday counterparts. This is likely due to several factors and we speculate that access to diagnostic and interventional procedures maybe contingent on decreased staff and lower weekend coverage that may impact regular workflow.
Abstract 311: CRP is Probably Not Causal in Atherosclerosis
Authors: Spence J David , Cocker Myra S , Tardif Jean-Claude , Beanlands Rob S
Published: March 2022

Background:
C-reactive protein (CRP) is thought to predict increased cardiovascular risk. However, the question of a causal relation to atherosclerosis is in doubt. We studied the relation of CRP to carotid total plaque area (TPA) and stenosis, and to plaque inflammation.

Methods:
In 527 patients with measurement of cardiovascular risk factors and CRP, linear regression was used to assess the relation of CRP to total plaque area (TPA) and stenosis. In a subset of patients scheduled for carotid endarterectomy, preoperative PET/CT with 18F- FDG was used to assess plaque inflammation, validated in endarterectomy specimens.

Results:
Mean age (SD) was 63.3 (12.27); 44.4% were female, 12.7% diabetic. 4.4% of patients had no plaque; in the rest it ranged from 1 to 788 mm2; median 102 mm2, interquartile range (IQR) 215 mm2. TPA > 361 mm2 was present in 10% of patients. Internal carotid (ICA) stenosis >= 60% was present in 38.3%; 34 had occlusion of one ICA; 1 had bilateral occlusion. Mean (SD) percent carotid stenosis (sum of right + left ICA) was 69.11 (56.0)%. Median hsCRP was 2.7; IQR 3.7. In stepwise linear multiple regression significant predictors of TPA (R2= 0.367, p< 0.00001) were age, smoking pack-years (pk-yrs), systolic blood pressure (SysBP), antihypertensive medication (BPMed), sex, lipid medication (LipMed) (in order of significance); CRP was excluded with p=0.35. Significant predictors of stenosis (R2 =0.226, p< 0.00001) were age, LipMed, pk-yrs, BPMed, SysBP. CRP was excluded with p=0.963. In 20 endarterectomy specimens, the direct burden of inflammation, as quantified by CD68 immunohistology, correlated with maximum 18FDG uptake (r=0.716, p<0.001). There was no evidence of a correlation between CRP and CD68 (r=0.159, p=0.50), nor with maximum 18FDG uptake (r=0.238, p=0.31). Furthermore, FDG was more strongly correlated with CD68 than CRP (p=0.02.CRP was inversely related to the extent of fibrous tissue (r=0.549, p=0.034).

Conclusion:
CRP was not associated with plaque inflammation, TPA, or stenosis of the carotid arteries. CRP is probably not a causal factor in atherosclerosis.
Abstract 317: Observations Concerning Changes in Flow-Mediated Dilatation Versus Low Flow--Mediated Constriction
Authors: Lytvyn Yuliya , Liuni Andrew , Luca MaryClare , Gori Tommaso , Parker John D
Published: March 2022

Flow mediated dilatation (FMD) is an accepted, non-invasive measure of endothelial function. FMD measures the capacity of the endothelium to respond to a stimulus but does not assess resting vascular tone. Prior to the measurement of FMD, the radial artery constricts as blood flow to the hand is interrupted, a phenomenon termed low-flow mediated constriction (FMC). The mechanisms of FMC remain poorly understood, however this measure is complementary to FMD. For example, exercise leads to vascular stimulation with an acute reduction in FMD but a reciprocal increase in FMC. It was hypothesized that interventions, which modify vascular nitric oxide bioavailability and alter endothelial function, impact FMC in normal subjects. The first protocol examined the impact of continuous therapy with transdermal nitroglycerin (GTN) on FMD and FMC (n = 18). After 5-7 days of GTN therapy, radial artery size was larger than before GTN (P < 0.01), FMD was reduced (P < 0.02, fig) but FMC was unchanged. In the second protocol (n = 8), vascular responses were assessed during sustained GTN therapy and 2 hours after GTN removal. FMD was blunted in the presence of GTN and remained blunted 2 hours after GTN removal (P < 0.01, fig), when vessel diameter had returned to baseline, but FMC was unchanged. In a third protocol (n = 14), the effect of inhaled salbutamol on vascular function was examined. Following stimulation with salbutamol, FMD was depressed (P < 0.02, fig) but FMC was not changed. These observations demonstrate that FMC does not change in response to sustained therapy with GTN, an intervention associated with marked abnormalities in endothelium dependent vasomotor responses, nor in response to beta-2 adrenergic receptor stimulation, an intervention known to acutely modify endothelial function. Both interventions are felt to mediate their effects through modification of nitric oxide bioavailability. Therefore, these findings confirm that FMC is not primarily dependent on nitric oxide.
Abstract 321: A Biomimetic Approach to Developing Antithrombotic Small-Caliber Prosthetic Vascular Grafts
Authors: Torres Melodie I , Onyejekwe Ifeanyi U , Finkelstein Eric B , Maier Kristopher G , Gahtan Vivian , Mather Patrick T
Published: March 2022

Introduction:
Prosthetic grafts smaller than 6 mm in diameter are linked with a high incidence of thrombosis and neointimal hyperplasia. We developed a novel graft material that provides controlled release of nitric oxide (NO), an antithrombotic agent that also promotes endothelialization within the lumen. Our very recent study focused on NO release kinetics from a similar composite that demonstrated controlled NO release for up to 200 d with a total release of 1.03 ± 0.09 μmol/mg.

Methods:
Grafts were fabricated using an electrospinning process that yields a micro-fibrous web composed of poly(ε-caprolactone) (PCL) . These fibers were doped with NO donor diethylenetriamine diazeniumdiolate (DETA/NO) and embedded within a polydimetheylsiloxane (PDMS) elastomer, which was found to regulate water transport and NO release rate.
In vitro
studies were tested in PBS at 37 C using a Griess assay to compute NO release. The shape memory properties inherent to this composite were used to form kink resistant acute angles through exposure to specific thermal cycles under strain.

Results:
NO release was proportional to DETA/NO doping and regulated by PDMS. Our present study revealed that the critical radius of curvature, below which kinks form, is a function of graft wall thickness and that a radii of curvature as small as 6 mm can be achieved using our unique process. The accompanying figure shows a 4 mm diameter graft with 0.8 mm wall thickness formed into a kink-free form with a radius of curvature of 6.7 mm.

Conclusions:
Incorporation of DETA/NO into a PCL/PDMS composite offers tunable long-term NO release and is a candidate for kink-resistant vascular grafts.
Abstract 323: Can Edaravone Protect From Kidney Damage Caused by Myonephropathic Metabolic Syndrome in Rats?
Authors: Yamamura Mitsuhiro , Mitsuno Masataka , Tanaka Hiroe , Ryomoto Masaaki , Fukui Shinya , Miyamoto Yuji
Published: March 2022

Introduction & Hypothesis:
Free radicals have been implicated in myonephropathic metabolic syndrome (MNMS), which damages not only muscles but also kidneys. At ATVB 2012 & 2013, we reported the free radical scavenger, edaravone (Radicut®, Mitsubishi Tanabe Pharma Co., Japan) suppresses muscle injury by MNMS. In this study, we evaluated whether edaravone can suppress kidney damage caused by MNMS.

Methods:
Lewis male rats (508 ±31 g, n = 10) were intraperitoneally injected with either 3.0 mg/kg of edaravone (edaravone group; n =4), or saline (MNMS group; n = 6). The MNMS models were induced, by clamping the bilateral common femoral arteries for 5 hours and then de-clamping. Five hours after de-clamping, the both kidneys were harvested and stained with hematoxylin & eosin. Normal kidneys were also harvested as control (n = 3). Kidney damage was evaluated by counting the number of cells in glomerulus (glomerular infiltration) and the density of residual tubular cells (enlargement of tubular cells).

Results:
The number of cells in glomerulus was significantly increased in MNMS group than that in the control (77.2 ±3.7 vs. 49.6 ±2.8 cells/glomerulus, p <0.01). However, the number of cells in glomerulus was alleviated in edaravone group (62.2 ±2.4 cells/glomerulus, p <0.01). The density of residual tubular cells was significantly decreased in MNMS group than that in the control (555 ±24 vs. 957 ±44 cells/mm2, p <0.001). The density of residual tubular cells was maintained in edaravone group (654 ±38 cells/mm2, p =0.06).

Conclusions:
MNMS was associated with significant glomerular infiltration and the enlargement of tubular cells. Edaravone may alleviate these kidney damages caused by MNMS.
Abstract 326: Microvascular and Arterial Remodeling Induced by Chronic Hypoperfusion
Authors: Choi Hye-Yeon
Published: March 2022

Background:
Atherosclerotic stenosis may induce chronic hypoperfusion of the brain when it is severe. Extremely high degree of stenosis which could cause cerebral hypoperfusion was reported to have a paradoxically lower risk of stroke development and less severe stroke when it develops. In the previous study, vascular remodeling and ischemic tolerance induced by chronic hypoperfusion were suggested as the probable mechanisms. However, it remains uncertain which type of vascular remodeling process, that is arteriogenesis or angiogenesis, occurs and from when it is observed after induction of cerebral hypoperfusion if it presents. This study was aimed to document temporal course of vascular remodeling and molecular expression related to arteriogenesis and angiogenesis after induction of sublethal cerebral hypoperfusion.

Methods:
Cerebral hypoperfusion was induced in wistar rats with the bilateral common carotid artery (CCA) ligation. Regional cerebral perfusion (rCP) was measured in the territory of the middle cerebral artery by laser Doppler flowmetry (LDF). Latex angiography was performed. Protein expression was examined by the western blot.

Results:
The rCP was decreased by 40.2% immediately after bilateral CCA ligation, when compared with the baseline, and recovered to the baseline level 14 days later. Increased arterial diameter, arterial tortuosity, and increased vascular density were observed at 1 week after bilateral CCA ligation. Connexin 37 was also increased at 1 week and decreased to the baseline level at 3 weeks, after bilateral CCA ligation. The levels of vascular endothelial growth factor and fibroblast growth factor-2 were not changed.

Conclusion:
In the vascular remodeling induced by sublethal cerebral hypoperfusion, arteriogenesis appeared to play a key role and to start within 1 week.
Abstract 33: Deficiency of Nogo-B Receptor Promotes the Nuclear Translocation of Liver X Receptor Alpha and Hepatic Lipogenesis
Authors: Hu Wenquan , Zhang Wenwen , Rana Ujala , Bordas Micelle , Zhao Baofeng , Han Jihong , Miao Qing (Robert)
Published: March 2022

The Nogo-B receptor (NgBR) was identified as a receptor necessary for Nogo-B induced morphogenesis of endothelial cells and developmental angiogenesis in Zebrafish. NgBR also is essential for the stability of NPC2 as well as NPC2 dependent cholesterol trafficking under serum-free condition. Here, we reported the novel roles of NgBR in regulating hepatic lipogenesis in vivo via NPC2-independnet pathways. Our results showed that either NgBR knockdown in hepatocyte under serum presence condition in vitro or NgBR knockout in mouse liver in vivo do not decrease NPC2 levels or increase NPC2-dependent intracellular cholesterol amount. However, NgBR deficiency resulted in significant increase of free fatty acid and triglyceride in hepatocyte in vitro and in mouse livers in vivo. Mechanistically, NgBR deficiency induces the nuclear translocation of the liver X receptor alpha (LXRα) so as to increase the expression of its downstream target genes involved in fatty acid synthesis, such as the sterol regulatory element binding protein-1 (SREBP-1) and fatty acid synthesis (FAS). Liver X receptor alpha (LXRα) is a member of nuclear receptor superfamily and one of master regulators for lipid metabolism. Real-time PCR results further showed that NgBR deficiency increased expression of LXRα-regulated gene such as ABCA1, SCD, ABCG8, SREBP1 and FAS, which are involved in lipid efflux and metabolism. LXRα knockdown by transient transfection of LXRα siRNA abolished NgBR deficiency caused accumulation of free fatty acid and triglyceride in stable hepatic cell lines and in the livers of NgBR knockout mice fed with the high fat diet. These observations demonstrated that NgBR deficiency induces lipid accumulation via the activation of LXRα pathway. It suggests that NgBR is essential for preventing hepatic steatosis.
Abstract 334: Intracoronary Allogeneic Mesenchymal Stem Cells and Cardiosphere-Derived Cells Exert Comparable Effects on Myocyte and Capillary Proliferation Without Promoting Functional Arteriogenesis in Swine With Chronic Myocardial Ischemia
Authors: Weil Brian R , Suzuki Gen , Leiker Merced M , Palka Beth A , Fallavollita James A , Canty John M
Published: March 2022

Objective:
Allogeneic icMSCs and icCDCs each improve function of hibernating myocardium but the underlying mechanisms remain unclear. We performed the present study to assess the effects of icMSCs and icCDCs on myocardial perfusion and remodeling of the coronary microcirculation in a large animal model of chronic regional ischemia.

Methods:
Cyclosporine-treated swine (200 mg/day) with hibernating myocardium from a chronic LAD stenosis received ~35 x 10
6
icMSCs, ~35 x 10
6
icCDCs, or vehicle. Regional function (echocardiography), adenosine-vasodilated perfusion (microspheres) and vascular remodeling were assessed 1-month after treatment.

Results:
Baseline LAD wall thickening (%WT) was reduced in all animals
(
LAD 38 ± 2% vs. 83 ± 5% in remote, p<0.01) but not different between treatment groups. Coronary flow during vasodilation was also critically reduced at baseline (LAD 2.4 ± 0.3 ml/min/g vs. 4.6 ± 0.3 ml/min/g in remote, p<0.01). Intracoronary stem cell infusion improved LAD %WT but did not alter vasodilated perfusion (Table). Although capillary density increased in relative proportion to the increase in myocyte number, there were no significant changes in arteriolar density following cell therapy.

Conclusion:
These data demonstrate that the functional improvement afforded by allogeneic icMSCs and icCDCs is accompanied by concomitant capillary angiogenesis and myocyte regeneration in hibernating myocardium. However, myocardial perfusion deficits persist due to an inability of either cell type to promote functional arteriogenesis, indicating that cell therapy-mediated functional benefits occur independently of changes in coronary blood flow.
Abstract 338: Akt-Mediated Phosphorylation of EBP50 Regulates Skp2 Stability and Vascular Smooth Muscle Cell Proliferation
Authors: Song GyunJee , Fitzpatrick Jeremy , Pellegrini Maria , Mierke Dale F , Bisello Alessandro
Published: March 2022

The PDZ domain-containing scaffolding protein, Ezrin-Radixin-Moesin-Binding Phosphoprotein 50 (EBP50) increases vascular smooth muscle cells (VSMC) proliferation and vascular remodeling following endoluminal vessel injury. Indeed, neointima formation is significantly reduced in EBP50 knockout mice. The mitogenic effect of EBP50 derives from the Akt-dependent stabilization of the S-phase kinase associating protein Skp2. However, the mechanisms by which Akt regulates EBP50-mediated cell proliferation are not known. We hypothesize that phosphorylation of EBP50 by Akt promotes its interaction with Skp2. This causes cytoplasmic re-localization and stabilization of Skp2 and VSMC proliferation.

Using Akt inhibitors and siRNA inhibition of Skp2, we show that the mitogenic effect of EBP50 in VSMC is dependent on both Akt and Skp2. In vitro and in vivo phosphorylation assays show that Akt phosphorylates EBP50 at T156 in the second PDZ domain. Interestingly, NMR, fluorescence polarization and co-immunoprecipitation experiments show that Skp2 interacts with the first PDZ domain of EBP50, suggesting that T156 phosphorylation exerts allosteric effects on the interaction with Skp2. Expression of EBP50 in primary VSMC induces a re-localization of Skp2 from the nucleus to the cytoplasm, leading to its stabilization and VSMC proliferation. These effects were abrogated by mutations within the first PDZ domain or of T156 to Alanine. Notably, the interaction with EBP50 does not directly affect the function of Skp2 because mutation of the C-terminal Leu to Ala, which abrogates the interaction with EBP50, does not decrease the ability of Skp2 to degrade p21cip1 and p27kip1. Consistent with the observations in primary cells, the expression of Skp2 in injured femoral arteries was reduced in EBP50 knockout mice compared to WT mice.

In conclusion, our studies identify a novel mechanism for the Akt-dependent regulation of cell proliferation by EBP50 during vascular remodeling. They indicate that inhibition of EBP50 may be useful in preventing neointima formation following angioplasty.