bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 268: Adiponectin Ameliorates Palmitate Acid Induced Endothelial Inflammation in Endothelial Cells
Authors: Chen Xiuping , Zhao Wenwen , Zhang Xuenong , Wu Chuanhong
Published: July 2021

Endothelial dysfunction (ED) is considered an early event of cardiovascular diseases including hypertension, atherosclerosis and so on. Inflammation participates centrally in all stages of cardiovascular diseases and is considered as a hallmark of endothelial dysfunction. In this study, the effect of adiponectin (APN), an adipocytokine derived mainly from adipocytes, on palmitate acid (PA)-induced inflammation in endothelial cells. Human umbilical vein endothelial cells (HUVECs) were treated with PA with or without APN pretreatment. The mRNA expression of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and ICAM-1 were measured with RT-PCR. The protein expression of ICAM-1, NOX1, NOX2, NOX4, and phosphorylation of MAPKs (JNK, ERK, and p38MAPK), IKKβ, p65 NF-κB were determined by Western blotting. Intracellular reactive oxygen species (ROS) and nitric oxide (NO) formation were determined with DCFH2-DA and DAF-FM respectively. APN significantly ameliorated PA-induced mRNA expression of TNF-α, IL-6 and ICAM-1 and protein expression of ICAM-1, NOX2, and phosphorylation of IKKβ, p65 NF-κB, p38MAPK, without affecting NOX2 and phosphorylation of JNK and ERK. APN also partly reversed PA induced ROS formation and NO decrease. NAC, a ROS scavenger, showed similar activities. The p38MAK inhibitor, SB203580, also reversed PA induced protein expression of ICAM-1 and mRNA expression of TNF-α, IL-6 and ICAM-1. Taken together, these results showed that APN improved PA induced endothelial dysfunction by regulating ROS/p38MAK/NF-κB pathways.

Acknowledgement: This study was supported by the National Natural Science Foundation of China (No. 81160048) and the Science and Technology Development Fund of Macau Special Administrative Region (No. 021/2012/A1).
Abstract 288: Role of Cilostazol in Preventing Instent Restenosis Among Patients With Peripheral Arterial Disease: A Meta-analysis
Authors: Iftikhar Omer , Oliveros Karla , Tafur Alfonso , Casanegra Ana
Published: July 2021

Objective:
Peripheral arterial disease (PAD) is associated with two to six fold increase in the cardiovascular mortality. Revascularization is indicated to relieve life limiting ischemic symptoms and improve wound healing. Primary patency for balloon angioplasty has been reported to be around 40 to 60% in the first year[8]. Stents have improved rates of primary patency but long-term patency rates are not comparable to bypass surgery, with many patients at high risk of in-stent restenosis. Many adjunctive therapies have been proposed to reduce the high restenosis rate. Our aim is to evaluate the use of cilostazol to prevent in-stent restenosis among patients with lower extremity arterial stenting.

Methods:
We performed a MEDLINE and EMBASE search, and reviewed the abstracts and manuscripts following the PRISMA guidelines. Patency rate after stenting was the primary efficacy outcome. At least 2 abstractors reviewed the study list and selected manuscripts. We calculated Q statistic and a homogeneity formal test. The odds ratio (OR) estimates were pooled by using the Mantel-Haenszel random-effects method. Data were analyzed using the R META package

Results:
We identified 524 studies, 20 articles were fully abstracted and 4 included in the metaanalysis. The total number of patients was 2434. The cilostazol and control groups were evenly divided. All studies were of moderate quality. Clinical characteristics including age and BMI were similar in the two groups. Stents were placed to treat de novo lesions. Two of the studies compared cilostazol to ticlopidine.

Cilostazol group patients had better primary patency rates after endovascular therapy than patients not taking cilostazol (OR 0.55, 95% CI 0.43 - 0.71). Heterogeneity was moderate with I2 of 38% and of moderate clinical relevance not statistically significant thus random effect model was kept. Omitting a single study did not affect the overall odds ratio of the other studies. Funnel plot suggested no publication bias.

Conclusions:
In stent stenosis among revascularized patients with PAD was 45% lower for patients who were on cilostazol.
Abstract 29: PCSK9 Association With Lipoprotein(a)
Authors: Tavori Hagai , Giunzioni Ilaria , Yeang Calvin , Tsimikas Sotirios , Shapiro Michael D , Duell Barton P , Fazio Sergio
Published: July 2021

Monoclonal antibodies (mAb) against PCSK9 lower plasma LDL-cholesterol levels by increasing the lifespan of LDL receptors (LDLR). Significant effects on lipoprotein(a) [Lp(a)] have also been observed, but the mechanism for this is neither known nor intuitive since Lp(a) is not cleared by LDLR. Our and other laboratories have shown that PCSK9 associates with LDL in plasma. Here, we aimed to study whether PCSK9 also associates with Lp(a) particles and whether this association depends on Lp(a) levels and/or apo(a) size. Using sandwich ELISA we determined that plasma PCSK9 is indeed associated with Lp(a) particles in patient with high Lp(a) levels (>30mg/dl). We then isolated LDL and Lp(a) fractions from plasma of 8 patients with high Lp(a) levels, ranging from 36 to 224 mg/dl, and determined PCSK9 and apoB levels as well as apo(a) isoforms. Our results show a 17.8 fold higher PCSK9/apoB ratio for Lp(a) compared with LDL (Fig. 1A), which suggests a preferential distribution of PCSK9 with Lp(a), at least in high Lp(a) individuals. Surprisingly, the preferential association of PCSK9 with Lp(a) was inversely correlated to Lp(a) levels (Fig. 1B) and independent of LDL levels. Since Lp(a) levels are inversely correlated with the size of apo(a), we set out to determine whether PCSK9 association with Lp(a) is related to the molecular weight of apo(a). Using plasma of patients with two distinct apo(a) isoforms, we show that PCSK9 associates exclusively with the higher molecular weight of apo(a) (Fig. 1C).

Our results suggest that Lp(a)-bound PCSK9 exists in plasma of patients with high Lp(a) and that this association depends on the size of apo(a). This provides a possible mechanism for the reduction in Lp(a) caused by PCSK9mAb, as immune complexes mAb-PCSK9-Lp(a) may be cleared via the mononuclear phagocyte system.
Abstract 292: Microparticle-encapsulated miRNAs Are Associated With Peripheral Artery Disease
Authors: Mitchell Adam , Gray Warren , Hayek Salim , Rooney Kim , Awad Mosaab , Quyyumi Arshed , Searles Charles
Published: July 2021

miRNAs are short, noncoding RNAs that circulate in plasma within membrane-bound vesicles, such as microparticles (MPs), or bound to proteins or lipoproteins. Circulating miRNAs are putative biomarkers for a wide range of diseases, including cardiovascular disease. Our group and others have evidence to suggest MP-encapsulated miRNAs, rather than total plasma miRNAs, may harbor more specific and functionally relevant biomarkers. We tested the hypothesis that MP-encapsulated miRNA levels would differ between patients with mild or severe peripheral artery disease (PAD) and healthy controls matched for age, gender, and race. Banked plasma stored at -80°C was obtained for 5 healthy subjects, 6 patients with mild PAD (ABI 0.8-1.0), and 10 patients with severe PAD (ABI < 0.4). MPs were isolated using a standard differential centrifugation protocol and quantified by flow cytometry. Four miRNAs implicated in vascular disease (miR-126-3p, miR-126-5p, miR-21, and miR-222) were quantified with qRT-PCR. Relative expression for each miRNA (-2ΔCt) was calculated and divided the total number of MPs to determine miRNA expression per MP. There was a non-significant trend towards increased levels of MPs in patients with mild (19,462 MPs/μl ± 5,213) and severe PAD (21,688 MPs/μl ± 7,175) compared to healthy controls (7244 MPs/μl ± 919). MP levels of miR-126-5p were increased among healthy subjects compared to patients with mild and severe PAD (p = 0.003), while MP miR-126-3p levels were significantly lower in healthy subjects (p = 0.002). miR-21 and miR-222 did not differ significantly between groups. None of the four miRNAs distinguished mild versus severe PAD. In conclusion, our data suggests that MP-mediated transport of miR-126-5p and miR-126-3p is dysregulated in the setting of mild and severe vascular disease, and supports the concept that the plasma MP fraction may harbor disease-associated miRNA profiles.
Abstract 299: Nitric Oxide Synthase Inhibition Attenuates the Formation of Notch-mediated Brain Arteriovenous Malformations
Authors: Wang Rong A , Huang Lawrence , Nystoriak Matthew A , Navedo Manuel F
Published: July 2021

Background:
Arteriovenous (AV) malformations (AVMs) are characterized by abnormal AV shunts that displace intervening capillaries. Brain AVMs (BAVMs) may cause life-threatening strokes and have limited treatment options. Mechanisms underlying AVM pathogenesis remain poorly understood, hindering therapeutic development.

Rationale:
We reported that endothelial expression of constitutively active Notch4 (Notch4*) in mice initiates BAVMs de novo through enlargement of microvessels without an increase in endothelial cell number or proliferation. Thus, we hypothesized that Notch4* initiates BAVMs by disrupting normal vasodilation and vascular tone. Nitric oxide (NO) regulates vasodilation and vascular tone, and Notch activation induces NO synthesis. Thus, we further hypothesized that Notch4* disrupts NO signaling, thereby permitting vessel enlargement and AV shunting.

Results:
Cerebral arteries isolated from Notch4* mutant mice were less constricted and exhibited decreased arterial tone than that of controls. Administering the NO synthase (NOS) inhibitor NG-nitro-L-arginine (L-NNA, 30 mg/kg, 1x/day) attenuated Notch4*-induced AV shunt initiation: AV connection diameter in L-NNA-treated Notch4* mutants was decreased compared to that of saline-treated Notch4* mutants (7.0±1.1 vs. 15.1±2.3 μm) at postnatal day 12, when vessel enlargement and AV shunting first become apparent. Although L-NNA treatment attenuated AV shunt initiation, it did not prevent AV shunt formation or its progression; LNNA-treated Notch4* mutants still exhibited BAVM-associated pathologies and mortality. In moribund L-NNA-treated Notch4* mutants, AV connection diameter was decreased compared to that of saline-treated Notch4* mutants (18.9±6.5 vs. 30.6±8.2 μm), but remained ~4 fold that of genetic controls (~5 μm). A reduction in diameter in L-NNA-treated Notch4* mutants was accompanied by an increase in median survival time (30 vs. 25 days in saline-treated Notch4* mutants).

Conclusions:
Our results suggest that arterial dysfunction may contribute to Notch4*-mediated BAVM formation, and that inhibiting NOS attenuates BAVM formation. These data support a role for NO pathway in Notch4*-mediated BAVM formation.
Abstract 302: Coronary Endothelial Dysfunction Is Associated With Increased Risk of Venous Thromboembolism
Authors: Prasad Megha , Reriani Martin , McBane Robert , Lerman Lilach O , Lerman Amir
Published: July 2021

Introduction:
Venous thromboembolism (VTE) is a cause of significant morbidity and mortality. The vascular endothelium may be involved in the pathogenesis of both VTE and arterial thrombosis, as endothelial injury and dysfunction may disrupt normal anticoagulation mechanisms. We aimed to test the hypothesis that coronary endothelial dysfunction (CED) is associated with development of VTE in prospective follow-up.

Methods:
Coronary vascular reactivity was evaluated in 502 patients with non-obstructive coronary artery disease by administration of intracoronary acetylcholine during diagnostic angiography. Microvascular CED was defined as ≤50% increase in coronary blood flow (CBF) from baseline in response to maximal dose of acetylcholine. After median follow-up of 6.3 years (IQR 3.5, 10.7), patients were assessed by standardized questionnaire, phone-call and review of medical records for development of VTE.

Results:
Median age of the population was 53 years (IQR 45, 62); 68% were females. Hypertension was prevalent in 40.8%, diabetes in 8.4%, and hyperlipidemia in 58.3% of patients. Of 502 patients, 279 had CED. There were no significant differences in baseline characteristics including cardiovascular and VTE risk factors between patients with and without CED (p>0.05). Eighty-nine percents of patients who developed VTE had CED. Patients who developed spontaneous or provoked VTE both had significantly lower % change in CBF than patients who did not develop VTE (p<0.05) (Figure 1). In univariate analysis, microvascular dysfunction was associated with increased risk of VTE (OR 6.55 (95% confidence interval (0.81, 52.79); p=0.04).

Conclusion:
We found a seven-fold increased risk of VTE with CED, suggesting a link between the two. Endothelial injury disrupting vascular homeostasis may predispose patients to VTE.
Abstract 312: Amyloids of Oxidized Apolipoprotein A-I Induce Amyloid Fibril Formation in Intact Apolipoprotein A-I
Authors: Chan Gary K , Witkowski Andrzej , Cavigiolio Giorgio
Published: July 2021

Background:
Amyloid deposition in atherosclerotic plaques increases with aging. Although a correlation between arterial amyloid deposits and cardiovascular events is yet to be established, the high incidence of amyloids associated with aortic intima and with atherosclerotic lesions indicates that amyloid deposits may contribute to atherosclerosis progression. Remarkably, apolipoprotein A-I (apoA-I) is the main component of these amyloids. We previously demonstrated that oxidation of apoA-I methionines by myeloperoxidase, at a concentration similar to that produced by activated macrophages in atherosclerotic lesions, promotes apoA-I amyloid fibril formation. Furthermore, recent studies revealed a hundred-fold increase in the amount of lipid-free apoA-I in atherosclerotic arteries compared to normal arteries. Notably, this apoA-I is heavily oxidized. Thus in the atherosclerotic plaques, high concentration of lipid-free apoA-I and an oxidative milieu are favorable conditions for apoA-I amyloid formation.

Hypothesis:
We tested the hypothesis that amyloid fibrils constituted of oxidized apoA-I can transfer the dysfunctional phenotype to intact apoA-I.

Methods:
Pre-formed amyloid fibrils constituted of oxidized apoA-I were incubated with a 10-fold excess of intact apoA-I at 37 °C, pH 6.0, with continuous vortexing. Kinetics of amyloid fibril formation by the pool of intact apoA-I were derived by measuring Thioflavin-T (ThT) fluorescence over a 6-day period.

Results:
After a lag-phase of 24-48 h, fibril formation proceeded with typical sigmoidal kinetics and reached plateau levels after about 6 days. In control samples, in which intact apoA-I was incubated in the absence of pre-formed fibrils, no significant changes in ThT fluorescence were detected for the same time course.

Conclusions:
Oxidized apoA-I amyloid fibrils can catalyze the aggregation of a large excess of intact protein. This observation bears important pathophysiological implications.
In vivo
, a small amount of amyloid fibrils could be produced by oxidized apoA-I in specific microenvironments of the atheroma; when transferred to the surrounding tissues, these amyloid seeds could induce extended amyloid formation in the large available pool of lipid-free apoA-I.
Abstract 314: HIV infection iIduces Structural and Functional Changes in High Density Lipoproteins
Authors: Siegel Marc O , Sviridov Dmitri , Bukrinsky Michael , Fitzgerald Michael L
Published: July 2021

Coronary artery disease is a growing clinical problem in HIV-infected subjects. Increased risk of coronary events in this population has been linked to low levels of HDL, but the effects of HIV infection and anti-retroviral treatment (ART) on HDL structure and function are poorly characterized. Here, we determined the composition and function of HDL particles isolated from plasma collected from ART-naive and ART-positive HIV-infected patients and compared them to HDL from a convenience control group. Proteomic profiling revealed decreased paraoxonase (PON) 1 and PON 3 in HDL from both treated and untreated HIV-positive patients, and PON activity of HDL from HIV-infected subjects was significantly lower than in control group. Lipidomic profiling found sphingomyelin and ether-linked glycerophospholipid species in the HDL particles correlated positively with viral load and negatively with CD4+ T cell counts in ART-naive subjects. Consistent with analysis of lipids, the level of PLTP showed a significant positive correlation with viral load and negative correlation with CD4+ T cell counts in ART-naïve HIV-positive samples. Given the low PON1 and 3 levels in HDL from HIV-infected subjects, we tested for the level of oxidized phospholipids in the HDL particles from a subset of HIV+ ART-naïve and treated subjects with completely suppressed viremia. No significant differences in oxidized lipids were found between HDL from control and untreated or PI-treated subjects, but a significant increase was detected in the NNRTI-treated samples. Similarly, the ratio of oxidized LDL to total LDL was significantly increased in the NNRTI-treated samples. These results suggest that reduced PON levels in HDL of HIV-infected subjects do not translate to increased levels of oxidized lipids, however, low PON may contribute to increased oxidation in NNRTI-treated patients. Finally, a negative correlation between cholesterol efflux capacity of HDL and viral load in ART-naive HIV-infected group was detected. Thus, HIV infection associates with a number of both protein and lipid compositional changes in HDL particles. Moreover, HIV infection affects cholesterol efflux function of HDL, and this may increase risk of atherosclerosis in this patient population.
Abstract 319: Design of Novel Chimeras to Provide Insight Into Structure/Function Activity of Apolipoprotein E3 and Apolipoprotein AI
Authors: Lek Mark , Ibe Nnejiuwa , Beck Wendy , Bielicki John K , Weers Paul M , Narayanaswami Vasanthy
Published: July 2021

Apolipoprotein (apo) E3 and apoAI are exchangeable apolipoproteins that play a dominant role in regulating plasma cholesterol levels and are considered anti-atherogenic. ApoE3 (299 residues, ~34 kDa) serves as a ligand for the low density lipoprotein receptor (LDLr) family of proteins that mediate cellular uptake and clearance of plasma lipoproteins. The ability of apoE3 to function as a ligand for LDLr resides in the N-terminal domain (NT) of apoE3. ApoAI (243 residues, ~28 kDa) lowers cholesterol levels through its ability to promote cholesterol efflux from peripheral tissues such as macrophages, forming HDL, which transports cholesterol back to the liver. Residues located in the C-terminal domain (CT) of apoAI mediate this function of promoting cholesterol efflux. The objective of this study is to generate chimeric apolipoproteins composed of apoE3 and apoAI to better understand the role of the C-terminal tail of the parent proteins. Two chimeras were generated by “domain swapping”: apoE3-NT/apoAI-CT and apoAI-NT/apoE-CT. They were overexpressed in E. coli, isolated and purified. Western blot analysis revealed the presence of epitopes from both parent proteins. Circular dichroism spectroscopy revealed that the α-helical content of apoE3-NT/apoAI-CT and apoAI-NT/E-CT was 43 and 54 %, respectively, which is comparable to that of the parent proteins. Chemical denaturation was used to probe for changes in protein stability. Addition of CT of apoE3 to NT of apoAI conferred more stability to apoAI, while addition of CT of apoAI to NT of apoE3 had no significant effect on apoE3. The rate of apolipoprotein-induced conversion of DMPC vesicles to discoidal structures was followed to assess their lipid binding capability. ApoE3-NT/AI-CT displayed a rate constant that was ~10x higher compared to apoE3, while apoAI-NT/apoE-CT showed a rate constant similar to that of apoAI. Whereas apoE3-NT/apoAI-CT elicits the ability to bind to the LDLr, apoAI-NT/apoE-CT did not. Lastly, both chimeras promote ABCA1-mediated cholesterol efflux from J774 macrophages, with the efflux capability being 2-fold greater for apoAI-NT/apoE-CT. These results show that CT of apoAI can promote lipid binding of apoE3, while CT of apoE3 can improve cholesterol efflux ability of apoAI.
Abstract 320: Procollagen C-Endopeptidase Enhancer Protein 2 Enhances SR-BI Mediated HDL Cholesterol Uptake and Reduces Atherosclerosis in Mice
Authors: Pollard Ricquita D , Blesso Christopher N , Zabalawi Manal , Fulp Brian , Gerelus Mark , Lyons Erica L , Zhu Xuewei , Nuradin Nebil , Li Xiang-An , Francone Omar L , Sahoo Daisy , Thomas Michael J
Published: July 2021

Epidemiological studies have shown an inverse correlation between plasma high density lipoprotein (HDL) concentrations and cardiovascular disease risk. At variance with these observations, clinical trials that significantly raised plasma HDL-C levels did not have improved clinical outcomes, emphasizing the importance of understanding HDL function. Recently, a significant correlation has been reported between human procollagen c-endopeptidase enhancer protein 2 (PCPE2) single nucleotide polymorphisms and HDL. PCPE2, a 52 kDa glycoprotein found in the extracellular matrix enhances cleavage of C-terminal procollagen by bone morphogenetic protein 1. Mice lacking PCPE2 have elevated concentrations of enlarged plasma HDL, a phenomenon associated with defective cholesterol efflux. HDL synthesis depends on ABCA1-mediated lipid efflux to lipid-poor apoA-I balanced by cholesterol uptake through hepatic scavenger receptor class B type I (SR-BI). Our studies focused on investigating if the elevated concentration of enlarged plasma HDL in PCPE2 deficient mice was atheroprotective. PCPE2 deficient mice were crossed with LDLr
-/-
mice (SKO) giving LDLr
-/-
, PCPE2
-/-
(DKO) mice that had elevated HDL levels compared to SKO mice. Despite elevated HDL levels, we found that DKO mice had significantly more lipid and CD68+ infiltration into the aortic root, similar to that reported for LDLr
-/-
, apoA-I
-/-
mice that lack plasma apoA-I/HDL. Furthermore, DKO mice showed reduced HDL apoA-I fractional clearance and reverse cholesterol transport rates compared to SKO mice suggesting PCPE2 plays a significant role in HDL remodeling and/or cholesterol uptake by the liver. To test the effect of PCPE2 on SR-BI function we incubated
3
H-cholesteryl ether (
3
H-CE) enriched HDL from SKO and DKO mice with CHO cells overexpressing PCPE2. Compared to CHO control cells, overexpression of PCPE2 increased
3
H-CE HDL uptake that was independent of HDL particle origin. Western Blot analysis showed no difference in SR-BI expression between control and PCPE2 transfected cells, suggesting that PCPE2 enhanced SR-BI function and promoted HDL cholesterol ester uptake. We conclude that PCPE2 is atheroprotective and an essential component of the reverse cholesterol transport system.
Abstract 358: Targeting of Heparin Binding EGF-like Growth Factor (HB-EGF) Efficiently Inhibits Atherosclerosis
Authors: Lee Sangderk , Lee Richard , Graham Mark , Kim Seon-Gu , Yang Lihua , Rateri Debra , Berliner Judith A , Lusis Aldons J
Published: July 2021

Objective:
Heparin-binding EGF-like growth factor (HB-EGF) is a receptor ligand that regulates inflammatory gene expression in the human aortic endothelial cells. In this study, we examined the effects of targeting HB-EGF using an antisense oligonucleotide (ASO) in the development of atherosclerosis.

Methods and Results:
Control and HB-EGF ASOs were injected intraperitoneally (i.p.; weekly; Dose: 50 mg/kg/wk) for 12 weeks into LDLR deficient mice fed a fat-enriched diet (21% fat; 0.2% cholesterol). Compared with the control ASO injected group (N=13), the HB-EGF ASO injected group (N=15) had efficiently reduced atherosclerosis in the arch of aorta (p<0.0001). The HB-EGF ASO also significantly reduced plasma cholesterol concentration (p = 0.0397). We observed that the VLDL was consistently reduced in all HB-EGF ASO injected mice tested. In liver tissue, the HB-EGF ASO induced significant downregulation of gene expressions of the sterol synthetic pathway as determined by a microarray and DAVID functional analysis (adjusted p = 5.5 x 10-9)). The HB-EGF ASO also induced lipid droplet accumulation in liver tissues as determined by histological analysis with liver tissue sections.

Conclusions:
This study indicates that HB-EGF is a key mediator for atherosclerosis and the targeting of HB-EGF is an efficient approach to inhibit lesion formation in the aorta. Downregulation of the sterol synthesis pathway and reduction of VLDL particles in the blood may contribute to the protective effects of HB-EGF ASO.
Abstract 362: Ccn1 Induction and Its Role in the Neointima Formation-induced by Carotid Artery Ligation in Mice
Authors: Hsu Pei-Ling , Chen Jheng-Sin , Mo Fan E
Published: July 2021

Aim:
Atherosclerosis, characterized by neointima formation, occurs preferentially at areas of disturbed flow. Extracellular matrix protein CCN1 is upregulated at the site of atherosclerotic lesions in humans and in animal models. Our aim was to examine the expression of CCN1-regulated by disturbed flow and its role in neointima formation.

Methods and Results:
Ccn1+/lacZ mice, in which a lacZ reporter gene was inserted to be driven by Ccn1-promoter, were subjected to complete ligation of the left common carotid artery. Progressive reduction in luminal diameters of the ligated artery were observed using B mode ultrasound compared with the right common carotid artery. Ccn1 expression, assessed by whole-mount X-gal staining, was detected in the intima and the media of carotid arteries 3 days after ligation, and the staining intensified between 1-4 weeks in the neointima after ligation. Specifically, Ccn1 expression was observed in endothelial cells, smooth muscle cells, and macrophages in the neointima. To dissect the role of CCN1 in the development of arterial lesions, knock-in mice carrying the integrin α6β1-binding-deficient mutant allele Ccn1-dm were tested in the carotid artery ligation model. Compared with wild-type mice, Ccn1dm/dm mice displayed significantly less neointima after ligation with improved endothelial function, less endothelial apoptosis, and lower oxidative stress, suggesting that the arterial expression of CCN1 induces endothelial dysfunction through its receptor integrin α6β1.

Conclusions:
CCN1 is a critical pathophysiological regulator mediating neointima formation induced by disturbed flow-generated after carotid artery ligation. CCN1 and its receptor integrin α6β1 represent potential therapeutic targets for atherosclerosis.
Abstract 366: Gender Differences Exist in Carotid Arterial Plaque Composition Among Men and Women with Carotid or Coronary Artery Disease and Elevated ApoB Levels
Authors: Paramsothy Pathmaja , Hong Jaekyoung , Isquith Daniel , Hulphers Elizabeth , Bai Hua , Shadzi Pey , Neradilek Moni , Gill Edward A , Zhao Xue-Qiao
Published: July 2021

Introduction:
Postmortem coronary artery histopathology and post-surgical carotid endarterectomy studies demonstrate that women have less calcification and inflammatory cells, but more smooth muscle cells than men. Gender differences in atherosclerotic plaque composition are not well known. Yet, the age-adjusted incidence of stroke and myocardial infarction are higher in men than women.

Hypothesis:
We hypothesized that gender differences exist in carotid plaque composition (CPC), % lipid rich necrotic core (LRNC) and % wall volume (PWV), comparing living women and men.

Methods:
The CPC study is a prospective, randomized study evaluating the effect of 1) atorvastatin + placebo + placebo vs 2) atorvastatin + niacin ER + placebo 3) atorvastatin + niacin ER + colsevelam on CPC. Participants had coronary or carotid artery disease and ApoB levels ≥120 mg/dL. CPC was evaluated using MRI. Baseline PWV [(wall volume/total vessel volume) х 100%], a measure of plaque burden that adjusts for variation in artery size, and % LRNC volume (among slices with LRNC present) were evaluated. Statistical analysis used Wilcoxon rank sum test, chi-square, and multivariate linear regression.

Results:
There were 82 women and 118 men. Women vs. men were older, mean±SD age 58±8 vs. 55±8 yrs. (p=0.008), had higher HDL-C, 48±13 vs. 40±10 mg/dL (p<0.001), higher ApoA1 147±24 vs. 126±19 mg/dL (p<0.001), higher ApoE 5.5±3.4 vs. 4.5±1.6 mg/dL (p=0.04), lower % of prior myocardial infarction 22% vs. 52% (p=0.02) and higher % of metabolic syndrome 48% vs. 46% (p=0.01). There were no significant differences in ApoB levels, 123±32 vs. 120±29 (p=0.4). After adjusting for age, HDL-C (strongly correlated with ApoA1, r=0.89), ApoE, prevalence of myocardial infarction and metabolic syndrome, the men-women difference in PWV was small and statistically non-significant ([[Unable to Display Character: ∆]] 0.0%, 95% CI -2.5 to 2.6%, p=1.0). However, among participants with LRNC, men had more % LRNC than women ([[Unable to Display Character: ∆]] 5.9%, 95% CI 1.4 to 10.3%, p=0.01) after the same adjustments.

Conclusions:
Gender differences exist in CPC in that there is a higher volume of LRNC in men compared to women. Further studies are needed which delineate the mechanisms underlying the differences in prevalence of LRNC comparing men to women.
Abstract 369: Protein Kinase C Epsilon-dependent Nox Activation Mediates Resistin-induced Cardiovascular Disease
Authors: Raghuraman Gayatri , Zuniga Mary , Mochley-Rosen Daria , Zhou Wei
Published: July 2021

Background:
Adipokine resistin induces vascular smooth muscle cell (VSMC) dysfunction, implying a vital role in cardiovascular disease. In this study, we examined the in vivo and in vitro effects of resisin and investigated the underlying mechanisms involving protein kinase C epsilon (PKCε) and reactive oxygen species (ROS).

Methods:
After guide-wire injury of distal left common carotid artery, ApoE-/- mice fed with a western diet were treated with or without subcutaneous infusion of resistin in the presence or absence of PKCε inhibitor for 4 weeks. The progress of intimal hyperplasia was monitored using an ultrasound micro-image system. NADPH oxidase (Nox) activity in the mouse coronary arteries was measured using a chemiluminescence assay. Additionally, we treated human coronary artery smooth muscle with resistin in the presence or absence of PKCε and Nox-specific inhibitors to determine mechanistic and functional effects of resistin in VSMCs functions. Reactive oxygen species (ROS) production was analyzed using confocal microscopy.

Results:
Resistin significantly enhanced injury-induced intimal hyperplasia in our murine model, which was mitigated by the PKCε inhibitor. Nox activity was also substantially augmented in the injured artery in a resistin-PKCε dependent manner. On the cellular level, resistin-induced ROS production was time-dependent and mitochondrial ROS seemed to be the trigger for cytosolic Nox-induced ROS. Inhibition of Nox completely abolished resistin-exaggerated VSMC proliferation, migration and dedifferentiation. Consistently, inhibition of PKCε significantly reduced resistin-induced cytosolic ROS production, Nox activity, and VSMC dysfunction.

Conclusion:
This is the first study showing that resistin-induced VSMC dysfunction and intimal hyperplasia is mediated through PKCε-dependent Nox activation pathway. These findings suggest potential molecular targets for cardiovascular disease.
Abstract 377: Retrograde Shear Stress does not Alter Endothelial Function in Healthy Subjects
Authors: Shah Sooraj , Katz Stuart
Published: July 2021

Introduction:
Endothelial dysfunction is a precursor and plays a significant role in development of atherosclerosis. Retrograde flow and resulting retrograde shear stress has been shown to adversely affect the endothelial function but the results may be flawed due to experimental conditions. Change in the limb position might result in increase in retrograde flow and adversely affect endothelial function. We hypothesized that elevation of arm above the level of the heart will result in increased retrograde flow and reduced endothelial function as measured by flow mediated dilation (FMD).

Methods:
We enrolled 27 healthy subjects after obtaining informed consent. Endothelial function was measured by FMD in brachial artery before and after arm elevation with elbow support for 30 minutes in fasting state. Flow velocities were recorded and analyzed at baseline, 10 and 20 minutes after arm elevation and at the end of 30 minutes.

Results:
The mean retrograde flow velocity was 9.9 ± 7.75 cm/sec at baseline, which increased to 15.8 ± 6.56 cm/sec (p=0.0019) after arm elevation. Out of the 27 subjects 21 (78%) subjects had increase in retrograde flow (20% or more). The difference in FMD change between the groups with increased or no change in retrograde flow was statistically non-significant (p=0.4).

Conclusion:
In conclusion arm elevation for 30 minutes was associated with statistically significant increase in retrograde flow without significant change in endothelial function in normal healthy subjects. The change in endothelial function between subjects who had augmented retrograde shear and those who did not was statistically not significant.
Abstract 380: Vascular Pcsk9: A Mediator for Atherogenesis Independent of LDL Receptor
Authors: Sun Hua , Tan Michael , Teng Ba-bie
Published: July 2021

PCSK9 (Proprotein convertase subtilisin/kexin type 9) increases the LDL levels by binding to hepatocyte LDL receptors (LDLR) and subjects it to degradation. We show that PCSK9 regulates apolipoprotein B (apoB) production by inhibiting its degradation process via the autophagic pathway, irrespective of the presence of LDLR. In addition to the role of PCSK9 in promoting hyperlipidemia, we hypothesized that vascular-PCSK9 in endothelial cells (EC) plays a role in initiating atherogenesis, irrespective of the presence of LDL receptor.

Our laboratory has generated double knockout mice lacking both LDLR and Apobec1 (apoB mRNA editing enzyme), named LDb, Ldlr-/-Apobec1-/-. They have the lipoprotein phenotype mimics human with hyperlipidemia; elevated levels of VLDL and LDL with low levels of HDL. They develop atherosclerotic lesions spontaneously. To investigate the role of PCSK9 in atherogenesis, we deleted Pcsk9 gene from LDb mice to generate the triple knockout mice (named LTp, Ldlr-/-Apobec1-/-Pcsk9-/-). In comparison to LDb mice (n=14), the LTp mice (n=8) had significantly decreased levels of cholesterol (387±10 vs. 313±14 mg/dl; p<0.0008) and triglyceride (304±15 vs. 204±2.3 mg/dl; p<0.0002). However, despite their high cholesterol levels at over 300 mg/dl, the atherosclerotic lesions in LTp mice were significantly decreased in comparison to LDb mice (8.8%±3.5 vs. 24%±3.3, p=0.004, n=5 vs. 5). We hypothesized that vascular PCSK9 regulates the development of atherosclerosis. We incubated LDL containing PCSK9 (LDL/PCSK9) on primary aortic endothelial cells (EC) obtained from LDb or LTp to study the effects of LDL/PCSK9 on inflammation. We show that LDL/PCSK9 could not induce the expressions of Lox-1, TLR-2, or ICAM-1 in EC from LTp, resulting in absence responses on proinflammatory markers (CCL2 and CCL7) and autophagic molecules (p62 and TRAF6). In conclusion, our results suggest that vascular PCSK9 play an essential role in atherogenesis.
Abstract 39: Circulating MiRNA Signature of Pet-Positive Abdominal Aortic Aneurysms: New Potential Predictors of Rupture
Authors: Courtois Audrey , Nusgens Betty , Hustinx Roland , Defraigne Jean-Olivier , Colige Alain , Sakalihasan Natzi
Published: July 2021

Prediction of abdominal aortic aneurysm (AAA) rupture by specific biomarkers is a challenging issue in the field of vascular diseases. Small non-coding RNAs, miRNAs, are potent post-transcriptional regulators of gene expression and were found to be present in blood and to represent valuable biomarkers for a variety of diseases, notably in cancer. Recently, we showed that a positive uptake of 18F-fluorodeoxyglucose (FDG) in the aneurysmal wall observed by positron emission tomography (PET) was correlated with a higher instability of the wall which could lead to its rupture. Identifying circulating miRNAs correlated with a positive PET could help discriminating patients at higher risk of rupture.

The expression level of 372 miRNAs was evaluated by using the miScript PCR system (Qiagen) in the plasma of 45 AAA patients among whom 24 had no FDG uptake (A0) and 21 displayed a positive PET (A+). In a first approach, 4 pools of plasma samples from the A0 and the A+ groups were analyzed. 14 miRNAs were found significantly modulated (8 downregulated and 6 upregulated, p<0.05) in A+ as compared to A0 patients. The modulation of expression of 8 of these miRNAs was validated by qRT-PCR on the individual samples. Among the decreased miRNA, miR-204-5p has been previously found to be downregulated in the aneurysmal wall compared to healthy aortas. These 8 miRNAs found modulated in blood were then analyzed in the media of A+ wall at the FDG uptake site and compared to a negative site of the aneurysm from the same patient. Two of these 8 miRNAs, miR-125b-5p and miR-204-5p, were significantly downregulated in the FDG uptake site in 6 patients.

In conclusion, 8 circulating miRNAs might represent a new signature of PET+ AAA, at high risk of rupture. Moreover, 2 of them are significantly downregulated in the metabolically active aneurysmal wall prone to rupture. It is noteworthy that a validated target of miR-125b-5p is MMP13, a collagenase that we previously showed to be largely increased in the positive FDG uptake site of adventitia and that could be potentially involved in the ultimate degradation of collagen leading to aneurysmal rupture.
Abstract 4: Macrophage p62/SQSTM1 Ameliorates Atherosclerosis by Sequestering Inclusion Bodies and Mediating Mitophagy
Authors: Sergin Ismail , Bhattacharya Somashubhra , Stokes Carl J , Curci John A , Razani Babak
Published: July 2021

Protein and organelle turnover is critical for cellular homeostasis and is prominently mediated by autophagy. Disruptions in autophagy lead to accumulation of protein aggregates and dysfunctional organelles such as mitochondria. Recent evidence suggests that the chaperone protein p62 is a critical link for targeting polyubiquitinated protein aggregates/damaged mitochondria to autophagosomes for degradation. Herein we describe a p62-centric mechanism of handling protein aggregates and dysfunctional mitochondria in atherosclerosis. Macrophages deficient in autophagy (ATG5-/-) or rendered deficient by incubation with atherogenic lipids have significantly increased levels of p62. This coincides with 1) the accumulation of polyubiquitinated proteins co-localizing with p62 and present as cytoplasmic inclusion bodies, and 2) p62 co-localization with mitochondrial markers. Aortas from atherosclerotic (ApoE-/-) mice also have progressive and marked elevations in p62, polyubiquitinated proteins, and mitochondrial reactive oxygen species that predominantly co-localize with plaque macrophages, a process further exacerbated in the autophagy-deficient setting. The formation of cytoplasmic inclusions and maintenance of adequate mitochondrial function appears to be dependent on p62. Lipid-loaded p62-null macrophages show polyubiquitinated protein accumulation present in a diffuse/disrupted cytoplasmic pattern. These macrophages also develop larger dysmorphic mitochondria with increased polarization and decreased oxidative phosphorylation capacity. As a result, p62-null macrophages display apoptotic susceptibility to atherogenic lipids and increased IL-1β secretion likely through mitochondrial-dependent inflammasome activation. Consistent with our in vitro observations, mice with either whole-body p62-deficiency or transplanted with p62-deficient bone marrow show significantly increased atherosclerotic plaque burden and lesion complexity with increased apoptosis and necrotic cores. Taken together, these data demonstrate a previously unrecognized atheroprotective role for macrophage p62 by facilitating the formation of inclusion bodies and maintaining healthy mitochondria.
Abstract 409: CD163-TWEAK Interaction Regulates Tissue Regeneration After Ischemic Injury
Authors: Akahori Hirokuni , Karmali Vinit , Polavarapu Rohini , Lyle Alicia , Weiss Daiana , Shin Eric , Husain Ahsan , Naqvi Nawazish , Dam Richard V , Habib Anwer , Choi Cheol U , King Adrienne L , Pachura Kimberly , Taylor Robert , Lefer David J , Virmani Aloke F
Published: July 2021

Macrophages are an essential component of the response to ischemic injury and direct inflammation and its resolution necessary for proper tissue repair. However, a complete understanding of their exact roles in this process is lacking. CD163, a type I transmembrane glycoprotein, is a receptor for hemoglobin:haptoglobin complexes and is exclusively expressed on macrophages. Its extracellular portion circulates in the blood as a soluble protein of unknown function. Here we show that during hindlimb ischemia, soluble CD163 functions as a decoy receptor for TWEAK (tumor necrosis factor-like weak inducer of apoptosis), a secreted pro-inflammatory cytokine of the tumor necrosis factor family, to regulate TWEAK-induced activation of canonical NF-κB and Notch signaling necessary for myogenic progenitor cell proliferation. Mice with deletion of CD163 demonstrated transiently elevated levels of TWEAK, which activated of both canonical NF-KB and Notch signaling, and muscle satellite cell proliferation and tissue regeneration not limited to the site of injury. Our findings highlight a novel mechanism by which macrophages coordinate systemic tissue repair after ischemic injury through control of the pro-inflammatory cytokine TWEAK.
Abstract 41: Postnatal Deletion of Vascular Smooth Muscle Cell Transforming Growth Factor beta Receptor 2 in Mice Exacerbates Marfan-syndrome-associated Aortic Dilation
Authors: Wei Hao , Angelov Stoyan N , Hu Jie Hong , Dichek David A
Published: July 2021

Objectives:
Thoracic aortic aneurysms (TAA) are an important cause of cardiovascular death and are often part of an autosomal dominant syndrome (e.g., Marfan syndrome; MFS). The role of transforming growth factor beta (TGF-β) signaling in TAA is controversial. Excessive TGF-β signaling in aortic smooth muscle cells (SMC) is proposed to cause TAA formation; however, much data support a protective role for aortic SMC TGF-β signaling. We investigated the role of SMC TGF-β signaling in the development of MFS-associated TAA by superimposing SMC-specific deletion of
Tgfbr2
on MFS-related aortic pathology of
Fbn1
C1039G/+
mice.

Methods:
We crossed
Tgfbr2
flox/flox
mice with
Acta2
-CreERT2 mice (Tamoxifen-inducible Cre driven by the SMC-specific
Acta
2 promoter) to generate mice with inducible deletion of
Tgfbr
2 in SMCs. 4 groups of mice were studied: 1)
Fbn1
+/+
,
Acta2
-CreERT2
o/o
, Tgfbr2
flox/flox
mice received Tamoxifen (wild-type control); 2)
Fbn1
C1039G /+
,
Acta2
-CreERT2
o/o
, Tgfbr2
flox/flox
mice received Tamoxifen (MFS; controlled for Tamoxifen); 3)
Fbn1
C1039G /+
,
Acta2
-CreERT2
+/o
, Tgfbr2
flox/flox
mice received vehicle (MFS; controlled for
Acta2
-CreERT2
+/o
); 4)
Fbn1
C1039G /+
,
Acta2
-CreERT2
+/o
,
Tgfbr2
flox/flox
mice received Tamoxifen (SMC-
Tgfbr2
-/-
superimposed on MFS). All mice received Tamoxifen or vehicle at 6 wk of age; ascending aortic anatomy was assessed at 16 wk of age.

Results:
Compared to wild-type mice, both groups of MFS mice had significantly increased ascending aortic diameter (~30%; p<0.05; ANOVA). Both groups of mice with MFS alone (Tamoxifen- and
Acta2
-CreERT2
+/o
-controls) showed comparable ascending aorta dilation and similar prevalence of aortic intramural hematoma. Superimposing the SMC-
Tgfbr2
-/-
genotype on the
Fbn1
C1039G /+
genotype mice further increased both ascending aorta diameter (>40%; p<0.05; ANOVA) and intramural hematoma rate (~4-fold; p<0.05,
X
° test) compared to mice with MFS alone. Studies are under way to delineate the alterations of vascular SMC TGF-β signaling in these models.

Conclusions:
Our data suggest that loss of TGF-β signaling in vascular SMC exacerbates aortic pathology in MFS mice and that vascular SMC TGF-β signaling protects against TAA formation induced by fibrillin deficiency.
Abstract 418: Microfibrillar-Associated Protein 4 Deficiency Reduces Size and Incidence Rate of Angiotensin II Induced Abdominal Aortic Aneurysms in Mice
Authors: Kirketerp-Møller Katrine L , Stubbe Jane , Schlosser Anders , Kejling Karin , Møller Jesper B , Marcussen Niels , Hansen Pernille B , Shi Guo-Ping , Holmskov Uffe , Sørensen Grith L
Published: July 2021

Microfibrillar-associated protein 4 (MFAP4) is an extracellular matrix (ECM) protein primarily located in elastic arteries. It can bind elastin and collagen, and furthermore activate vascular cells through cellular integrin binding and modulate matrix metalloprotinase (MMP) activity. We hypothesized that lack of MFAP4 would decrease vascular inflammation and abdominal aortic aneurysm (AAA) formation.

AAA was induced in 9-11 week old mice using two experimental mouse models:

1) Male

Mfap4
-/-
/ApoE
-/-

double knock-out (dKO) and

ApoE
-/-

littermate control mice were feed western diet and subjected to continuously angiotensin II (AngII, 1000 ng/kg/min) infusion for 9-28 days via subcutaneous osmotic mini-pumps. Arterial blood pressure was measured in the femoral artery.

2) 1.5 U/mL elastase was infused into the infrarenal aorta in

Mfap4
-/-

and littermate

Mfap4
+/+

mice for 5 minutes. Aortic blood flow was restored and the mice recovered for 9-16 days.

Aortic diameter was measured in mice subjected to AngII or elastase infusion at day 28 and 16 respectively. MMP activity was detected by zymography.

No difference in AAA formation was observed between genotypes after elastase perfusion. In response to AngII infusion dKO mice showed a significantly decrease in AAA diameter and incidence rate compared to

ApoE
-/-

mice. AngII-induced increase in blood pressure was not dependent of MFAP4. However, there was decreased aortic arch atherosclerotic plaque formation, MMP2 and MMP9 activity in aortic tissue from dKO mice compared to

ApoE
-/-

mice. Furthermore there was a non-significant tendency of decreased elastin degradation score in the AngII infused dKO mice, however this was not observed in the elastase perfused mice. Activity of MMP12 and extent of infiltrating leukocytes in aneurysmal tissue from both models will be further investigated.

In conclusion we observed a decreased AAA formation and MMP activity in

Mfap4
-/-
/ApoE
-/-

mice which was not explained by variation in blood pressure or altered elastin degradation. The data suggest that MFAP4 induces MMP2-activity and thus the propensity for AAA formation.