bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 310: GLP-1 Cleavage Product (9-36)-NH2 Exerts Pro-survival Effects on Smooth Muscle Cells in Advanced Inflammatory Hemodynamic Conditions
Authors: Feaver Ryan E , Rekhter Mark , Walton Sandi , Day Nathan , Alsina-Fernandez Jorge , Kowala Mark , Wamhoff Brian , Blackman Brett
Published: March 2022

Glucagon-like peptide-1(GLP-1), an incretin hormone, has been shown to have atheroprotective effects on the cardiovascular system in animal models of atherosclerosis and diabetes, and in several clinical trials. These beneficial effects are presumed to occur through active GLP-1 (7-36)-NH2/(7-37), and its receptor GLP-1R; however, within minutes GLP-1 (7-36)-NH2/(7-37) becomes cleaved and inactivated by DPP4 into its smaller metabolite, GLP-1 (9-36)-NH2(9-37). It still remains unclear how GLP-1 (7-36)-NH2/(7-37), its metabolites, and GLP-1R provide beneficial vascular effects. We sought to elucidate the role of GLP-1 on vessel wall health using a novel in vitro human surrogate system that cocultures human endothelial (EC) and smooth muscle cells (SMCs) and applies human hemodynamics from the carotid bifurcation, a site prone to developing atherosclerosis, combined with other atherogenic factors (oxidized LDL, TNFα). Though, we found GLP-1R to be highly expressed in ECs and SMCs from both healthy and advanced inflammatory vascular environments, atheroprotective effects from the addition of GLP-1 (7-36)-NH2 or (9-36)-NH2 acted primarily on SMCs. In SMCs from atherogenic hemodynamic conditions, GLP-1 (7-36)-NH2 reduced NFkB activity and increased the anti-oxidant response protein, HO-1, via GLP-1R. A novel finding was that the cleaved metabolite, GLP-1 (9-36)-NH2, elicited the same response. Though GLP-1 (9-36)-NH2 did not alter downstream inflammatory gene expression, the NFkB-dependent pathways converged on pro-survival signaling since the metabolite GLP-1 (9-36)-NH2 uniquely decreased apoptotic signaling, including cleaved Caspase-3. These same responses were not seen with cleavage-resistant forms of GLP-1 (7-36)-NH2, but were rescued by adding back the cleavage product, (9-36)-NH2_further illustrating the distinct role of GLP-1 (9-36)-NH2 in promoting cell survival. This suggests future therapeutics would benefit by combining native GLP-1 along with its cleavage product, GLP-1 (9-36)-NH2. These findings demonstrate the novel effect of native GLP-1 and its cleavage-products on vessel wall health and provide new insights into creating more effective GLP-1 therapeutics for treatment of atherosclerosis and diabetes.
Abstract 314: The Safety of Preoperative Vardenafil in Patients Undergoing Coronary Bypass Surgery.
Authors: Ali Asghar , Binder Andrew , Mohmand Asad , Stewart Helen , Pipkin Margaret , Martin Lisa , Szentpetery Szabolcs , Katlaps Gundars , Jovin Ion S
Published: March 2022

Objectives

Advances in cardiac surgery have significantly improved the outcomes for patients with myocardial ischemia. However, ischemia/ reperfusion injury represents a mechanism that continues to limit the effectiveness of coronary bypass graft surgery (CABG). Phosphodiesterase 5 inhibitors have been shown to exhibit powerful cardioprotective effects in animal models of ischemia-reperfusion injury. We evaluated the safety of vardenafil in patients undergoing cardiac surgery.

Methods

10 male patients undergoing CABG with or without valve surgery were enrolled in the study. Subjects were given a onetime dose of 10 mg oral vardenafil on the morning of surgery. They were monitored for adverse events and left ventricular ejection fraction changes; pertinent clinical parameters were also recorded. Their postoperative parameters and outcomes were also compared with 47 patients who underwent CABG in the same period of time (control group).

Results

The study drug was tolerated well. There were no episodes of hypotension or deaths in the group receiving vardenafil. The left ventricular ejection fraction did not change significantly after surgery (0.54±0.08 vs 0.53±0.09; P=0.76) in the vardenafil group. The vardenafil group and the control group were similar in terms of clinical characteristics. The type of operation and operative parameters were also similar between the two groups. Peak troponinT levels were not significantly lower in the vardenafil group compared to the control group (9.1 ± 8.3 vs 12.5 ± 9.3 ng/mL; P = 0.26, respectively). The median duration of intubation was not significantly different in the vardenafil group compared to the control group (21.4 ± 10.1 vs 27.4 ± 15.2 h; P = 0.14). The length of hospital stay was also not significantly different between the vardenafil group and the control group (11.1 ± 13.2 vs 10.0 ± 4.7 days; P=0.80, respectively).

Conclusions

Vardenafil is well tolerated when given to patients immediately prior to CABG surgery and appears safe especially in the setting of the intensive care unit where blood pressure and other vital signs are closely monitored. A larger trial is needed to document the cardioprotective effects of the drug in these patients.
Abstract 327: Ankle-Brachial Index is Associated with Subendocardial Viability in Biracial (black-white) Young Adults: The Bogalusa Heart Study.
Authors: Fernández Alonso Camilo , Li Shengxu , Thakkar Badal , Srinivasan Sathanur R , Chen Wei , Blandon Roberto , Berenson Gerald S
Published: March 2022

Ankle-Brachial Index (ABI) has been widely used clinically to define peripheral artery disease (PAD). Previous studies have established the correlation between ABI and coronary artery disease among men and women; however, limited information is available on the association between ABI and subendocardial viability in terms or Subendocardial Viability Ratio (SEVR) -a measure of myocardial perfusion-, among asymptomatic biracial (black-white) young adults.

We assessed the hypothesis that ABI is directly correlated with SEVR in asymptomatic biracial (black-white) young adults.

As a part of The Bogalusa Heart Study, a bi-racial (black/white) community based study, noninvasive assessment of ABI and SEVR was obtained in 610 asymptomatic individuals aged 29-51years (mean 43.4; 68.7% white and 54.6% female), along with evaluation of traditional cardiovascular risk factors. The association of ABI with SEVR was tested through multivariable-adjusted linear regression.

Black and female participants had significantly lower mean levels of SEVR (p<0.001) and ABI (p<0.001), compared to their counterparts. In race-specific stepwise multiple regression analyses, with adjustments for age, body mass index, sex, smoking status and traditional CV risk factors, significant direct associations were observed between ABI and SEVR in both, white (B=0.25, p<0.001) and black participants (B=0.11, p<0.03), with a stronger association in white participants.

The observed association of ABI with SEVR in black-white asymptomatic adults supports the hypothesis that, structural changes in the peripheral vasculature are reflective of changes in the coronary arteries, even at subclinical level, which may aid to enhance early detection of coronary artery disease in asymptomatic individuals.
Abstract 341: Expression of Leukocyte CD39 in Acute Coronary Syndrome
Authors: Sutton Nadia R , Baek Amy , Liao Hui , Kanthi Yogen , Song Christopher , Visovatti Scott , Pinsky David J
Published: March 2022

Background

CD39 (ENTPD-1) is a membrane-spanning ectonucleotidase that is responsible for the phosphohydrolysis of ATP to ADP and ADP to AMP. As increased myocardial infarct sizes have been reported in murine CD39 knockout models when compared with controls, we hypothesized that CD39 might be expressed as part of the myocardial injury/repair response. CD39 levels have not been heretofore described in humans with acute coronary syndrome (ACS).

Objectives

To measure CD39 mRNA and protein levels in human study subjects with ACS compared with healthy controls.

Methods

Healthy subjects (n=24), defined as having no history of coronary or peripheral arterial disease, and subjects with ACS (n=13) were enrolled in the study. Blood was collected via venupuncture for healthy controls or at the time of cardiac catheterization for ACS subjects. Plasma and leukocytes were isolated from blood, and soluble CD39 protein from leukocytes was measured using ELISA. mRNA was extracted from leukocytes, and qPCR was used to measure differences in leukocyte CD39 mRNA. Mass spec was used to assess plasma ATP, ADP, AMP, and adenosine levels.

Findings

Increased CD39 mRNA expression was found in subjects with ACS when compared with healthy controls (2.39 vs. 1.66, p=0.021). There was a trend towards increased leukocyte CD39 protein concentrations in ACS patients (n=5) compared to controls (n=9) (7.50 ng/ml vs. 2.64 ng/ml, p=0.057) when measured by ELISA. There was a trend toward increased levels of soluble plasma CD39 levels in ACS patients (n=12) when compared with controls (n=24) (2.39 ng/ml vs. 1.67 ng/ml, p=0.59) when measured by ELISA. ACS patients (n=11) were found to have higher levels of circulating ADP as assessed by Mass spec when compared with healthy controls (n=17), 2.02 vs. 0.67, p=0.03. Leukocyte CD39 levels positively correlated with ATP (r
2
=.231) and ADP (r
2
=.155) levels (n=10).

Conclusions

This is the first report of increased CD39 gene expression in patients with ACS, when compared with healthy controls without known coronary disease. A trend toward increased plasma and leukocyte CD39 protein expression was present in those with ACS when compared with controls. CD39 could serve as a modulator of vascular homeostasis in ACS.
Abstract 353: Diabetes Induces Increased Expression of Matricellular Osteoblast Factor Periostin and Inhibits the Calcification Inhibitor Matrix Gla Protein (mgp) in Rat Aorta
Authors: Wajih Nadeem
Published: March 2022

It is increasingly recognized that diabetes is associated with increased risk of cardiovascular events and vascular calcification. To understand the mechanism of vascular calcification in diabetes we used a rat model of type 2 diabetes. Rats were fed
ad libitum
with diet containing 45 kcal % fat (soybean oil and lard with 0.95 mg cholesterol /gram of lard), 35 kcal % carbohydrates and 20% kcal protein (HF diet) for eight weeks, followed by a single low dose of stz (30mg/kg intra peritoneally). Rats were then allowed to developed diabetes for sixteen weeks. Average blood glucose in diabetic HF rats 517.6mg/dl vs 87.8mg/dl in HF rats. The expression and immunohistochemical changes were analyzed in aorta of these rats after sixteen weeks of diabetes. Conformational specific gamma carboxylated (Gla) and undercarboxylated (Glu) MGP antibodies were used to assess the expression of Gla and Glu MGP in control and diabetic HF rat aorta. Western blot analysis show significantly reduced expression of Gla MGP in diabetic HF rat aorta and higher Glu MGP expression compared to control rat aorta suggesting that diabetic conditions inhibit the gamma carboxylation of MGP in aorta, gamma carboxylation modification is necessary for the MGP to work as physiological calcification inhibitor. Similarly, immunohistochemical analysis of diabetic HF rat and control rat aorta show increased staining of Glu MGP than Gla MGP compared to control rat aorta further confirm our Western data that modification of Gla MGP is inhibited in diabetic HF rats. Furthermore, immuno-histochemical data also show significantly increased in diabetic HF rat aorta expression of Periostin a matricellular osteoblast specific factor expression compared to control rat aorta. Our findings suggest that active diabetes impair the physiological properties of calcification inhibitor Gla MGP in aorta and increases the osteogenic factors in diabetic HF rats. Increased expression of osteoblastic factor and inhibition of Gla MGP suggests that diabetic HF rat aorta is under active transformation to become more bone like, precursor of arterial calcification.
Abstract 356: Effect of Long-Term Diet-Induced Hyperglycemia and Hyperlipidemia on Placenta Growth Factor Expression in Mouse Heart
Authors: Silva Asitha T , Lloyd Pamela G
Published: March 2022

Arteriogenesis (collateral artery remodeling) is a vital adaptation of the vasculature in response to occlusion which serves to improve O2 and nutrient delivery to distal tissue, thereby decreasing risk of tissue damage due to ischemia. Stimulation of arteriogenesis would be highly beneficial in diabetics, who are especially prone to develop ischemic cardiovascular disease. However, arteriogenesis is suppressed in diabetes by an undefined mechanism. Placenta growth factor (PLGF) is a key arteriogenic factor; thus, we hypothesized that decreased PLGF levels might contribute to impaired arteriogenesis in diabetes. To test this hypothesis, we utilized three mouse models of long term (6 mo) diet-induced metabolic dysfunction: hyperglycemic + hyperlipidemic (high fat fed C57BL/6; HG/HL, n=7), hyperlipidemic (low fat fed ApoE-/-; HL, n=5) and extremely hyperlipidemic (high fat fed ApoE-/-; EXHL, n=12). A normoglycemic + normolipidemic control group (low fat fed C57BL/6; CONT, n=9) was also studied. Both males and females were studied to identify gender differences. Mouse phenotype was confirmed by plasma cholesterol assay, intraperitoneal glucose tolerance testing, and body weight. Cardiac PLGF gene expression was decreased in both male and female EXHL (~50%, p<0.01 and ~42%, p=0.09 respectively), compared to HL. PLGF mRNA was also decreased in male (~43%, p=0.06) but not female HG/HL, compared to CONT (n=5). This decrease was specific to PLGF, as mRNA for VEGFR1 (PLGF receptor) and the related growth factor VEGF-A was not affected by diet. We previously showed that PLGF is upregulated by low, physiological levels of hydrogen peroxide in vitro, suggesting that reactive oxygen species (ROS) signaling may be important in regulating PLGF. Since oxidative stress occurs in both hyperglycemia and hyperlipidemia, and arteriogenesis is known to be subject to a “redox window,” the inhibition of PLGF expression we observed may be mediated by oxidative stress. Plasma isoprostane levels confirmed that oxidative stress existed in HG/HL, HL, and EXHL compared to CONT. We conclude that hyperlipidemia is a key metabolic parameter influencing PLGF expression in cardiac tissue. Further studies to identify the role of oxidative stress in PLGF expression are needed.
Abstract 361: Characterization of Lipoproteins in the Hypertriglyceridemic Fredrickson-Levy Dyslipidemic Phenotypes with Chylomicrons: The Very Large Database of Lipids (VLDL-7)
Authors: Hassan Kamran , Jones Steven R , Martin Seth S , Joshi Parag S , Blaha Michael J , Elshazly Mohamed , Kulkarni Krishnaji R , Toth Peter P
Published: March 2022

Background

The major dyslipidemia (DL) classes (I-V) are relatively rare with limited phenotypic characterization across large populations.

Methods

We studied 1,340,614 U.S. adults from the Very Large Database of Lipids and identified those with chylomicron (CM) excess (types I and V). Lipid testing was by ultracentrifugation (VAP, Atherotech, Birmingham, AL). The distributions of total cholesterol (TC), HDL-C, LDL-C and triglycerides (TG) in the database were superimposable with those in the NHANES 2007-2008 survey. We used the following criteria to classify the excess lipoprotein components of DL in subjects with TG >130 mg/dL: CM defined by TG/Total cholesterol (TC) ratio > 9 only (Type I) and CM with >90th percentile VLDL-C (Type V). Subjects with types II, III, or IV DL were excluded.

Results

Type I DL was characterized by very high TG and very low HDL-C, LDLr-C, non-HDL-C and normal VLDL-C and RLP-C. Type V was characterized by TG approximately twice that of type I, low HDL-C and LDLr-C, and elevated VLDL-C, RLP-C and non-HDL-C. The VLDL3-C/IDL-C ratio was approximately 2, twice that of the non-CM groups (Table).

To our knowledge, this is the largest single report of subjects with these rare hyper-CM phenotypes. The findings support the mechanism of reduced CM clearance and impaired conversion of VLDL to IDL and LDL. The marked reduction in HDL-C is consistent with cholesterol ester transport protein mediated TG-Cholesterol ester exchange between CM and apoB100 lipoproteins.
Abstract 364: Loss Of Systemic C-kit Function Determines Atherosclerosis Burden In Hyperlipidemic Mice
Authors: Song Lei , Duque Juan Camilo , Pena Angela , Shehadeh Lina , Webster Keith A , Pham Si M , Vazquez-Padron Roberto I
Published: March 2022

Introduction

C-kit is one of the most important tyrosine kinase receptors dynamically regulated in healthy and diseased blood vessels. However, whether c-Kit signaling is beneficial or detrimental to the vasculature remains elusive. This study assesses the impact of the loss of c-Kit in somatic and circulatory cells on atherosclerosis.

Methods and Results

c-Kit expression was abundant in human aortas as determined by RT-PCR, Western blot and immunofluorescence (IF) confocal microscopy. The vascular expression of this receptor varied among patients and tended to decrease in atherosclerotic arteries. Interestingly, in the murine aorta from a c-Kit reporter mouse, GFP was only found in a sub fraction of quiescent VSMC that reside just below the endothelium. c-Kit deficiency favored VSMC proliferation when aortic explants from c-Kit mutant (W/Wv) and wild type (WT) mice were simultaneously placed in a tissue culture system (cells per well: 1337 ± 167 vs. 696 ± 108, p= 0.0123). To measure the contribution of c-Kit signaling to atherosclerosis disease in ApoE KO mice, we fed ApoE KO mice carrying the c-Kit mutant alleles W and Wv with a high-cholesterol diet for 16 weeks. The atherosclerosis plaque burden in these mice was 2.5 greater than in control ApoE KO kit
+/+
mice (affected proportion of the aorta: 0.25 ± 0.03 vs.0.104 ± 0.01, p= 0.0002). Finally, we rescued lethally irradiated ApoE KO W/Wv and control ApoE KO kit
+/+
mice with ApoE KO kit
+/+
bone marrow (BM) cells to determine how the lack of c-Kit activity in BM-derived cells modifies atherosclerosis. While BM transplantation ameliorated most of the hematopoietic defects in the ApoE W/Wv recipients, it had little or no effect on preventing atherosclerotic disease development (affected proportion of the aorta: 0.09 ± 0.01 vs.0.05 ± 0.007, p= 0.03).

Conclusion

Our results demonstrate for the first time that somatic c-Kit-positive cells may confer protection against atherosclerosis in hyperlipidemic mice.
Abstract 367: Macrophage 12/15-lipoxygenase Mediates Insulin-like Growth Factor I (IGF-1)-induced Suppression of Foam Cells: Potential Mechanism Responsible for IGF-1-induced Atheroprotection
Authors: Sukhanov Sergiy , Vaughn Charlotte , Snarski Patricia , Kim Catherine , Delafontaine Patrick
Published: March 2022

We have shown that IGF-1 infusion into Apoe-null mice suppressed atherosclerosis. To define mechanisms we performed RT-PCR array and found that IGF-1 (0.8 mg/kg, i.p, 7d) reduced aortic 12/15-lipoxygenase (LOX) expression (64±6% decrease vs. saline control, P<0.05). Macrophage LOX is a major pro-atherogenic enzyme that mediates oxidation of native low density lipoprotein (nLDL) and via this mechanism LOX promotes lipid uptake by macrophages (MΦ) and formation of foam cells. IGF-1 infusion into Apoe-null mice (1.5 mg/kg/d, 12 wks on a Western diet) reduced LOX plaque immunopositivity (74±9% decrease, P<0.05), suppressed plaque oxidative stress (64±7% decrease, IHC with 8-oxodG a/b, P<0.05), reduced plaque lipids (35%±6 decrease, Oil Red staining, P<0.05) and decreased atherosclerotic lesion area in aortic valve (IGF-1: 0.259 ± 0.018 mm2, saline: 0.356 ± 0.031 mm2; P<0.05).

To further define mechanisms THP-1-derived MΦ were exposed to IGF-1. IGF-1 (50 ng/ml, 16h) reduced LOX mRNA (49±6% decrease, RT-PCR, P<0.05), decreased LOX protein (42±6% decrease, immunoblotting with LOX a/b, P<0.05) and reduced LOX activity (32±5% decrease, 15S-HETE ELISA, P<0.05) and IGF-1 suppressed STAT6 transcription factor binding to LOX gene promoter region (CHIP assay with STAT6 a/b). IGF-1 markedly reduced oxidation of nLDL mediated by THP-1 or mouse peritoneal MΦ (85±6% and 67±5% decrease, respectively, MDA assay). IGF-1 decreased subsequent LDL uptake (i.e. formation of foam cells) mediated by THP-1 or WT peritoneal MΦ (45±8% and 51±3% decrease, respectively, Oil Red staining); however both lipid uptake and IGF-1 effect were decreased in peritoneal MΦ isolated from LOX-null mice (P>0.05). These data indicate that specific LOX downregulation mediates IGF-1-induced suppression of nLDL oxidation and formation of foam cells. In summary, IGF-1 reduced LOX expression in atherosclerotic plaque and this effect correlated with reduction in plaque lipids and in total atherosclerotic burden. IGF-1 downregulated LOX in cultured MΦ via inhibition of STAT6-dependent transcription and IGF-1 decreased lipid oxidation and uptake by MΦ. Taken together these data strongly suggest that MΦ LOX downregulation mediates IGF-1-induced reduction in foam cells and in atherosclerosis.
Abstract 37: Association between High-Density Lipoprotein Cholesterol Subfractions and Carotid Intima-Media Thickness
Authors: Tiozzo Eduard , Gardener Hannah , Hudson Barry I. , Dong Chaunhui , Weiss Danielle , Della Morte David , Elkind Mitchel S , Disla Norbelina , Wright Clinton B , Sacco Ralph L , Rundek Tatjana
Published: March 2022

Background

Recent trials have challenged the protective effect of HDL in atherosclerosis, implicating the need to shift the focus from total HDL concentration to its function and major subfractions. The objective of this cross-sectional study was to investigate the relation between HDL2 and HDL3 subfractions and carotid intima-media thickness (IMT), an early indicator of atherosclerosis and surrogate marker of ischemic stroke.

Methods

In NOMAS, a prospective population-based study of the incidence and risk factors of stroke, we evaluated 1041 participants (mean age 68±9 years; 38% men; 62% Hispanic, 19% black, 17% white) with available data on HDL subfractions and carotid IMT assessed by a high-resolution 2D carotid ultrasound. Carotid IMT was a composite measure of the mean IMT in all carotid artery segments. The cross-sectional association between HDL-subfractions and IMT was analyzed by multiple linear regression.

Results

We mean±SD of total HDL was 46.15±14.25 mg/dl (median = 44.00), HDL2 14.59±8.29 mg/dl (median = 13.00), HDL3 31.06±8.32 mg/dl (median = 32.00). The mean IMT was 0.90±0.08 mm. After controlling for demographics, LDL, triglycerides, and cholesterol-lowering medication, a strong association was observed between total HDL (per SD, beta=-0.008, p= 0.01), HDL2 (per SD, beta=-0.006, p=0.03) and HDL3 (per SD, beta=-0.007 p=0.01) in relation to IMT. A significant interaction was observed between diabetes (vs. non-diabetes) and HDL2 in relation to IMT (p=0.01; -0.018mm).

Conclusion

The observed strong independent inverse associations between HDL, HDL2 and HDL3 and carotid IMT. HDL2 was only significantly associated with IMT among individuals with diabetes. These observations show novel evidence for a potential role of HDL2 and HDL3 subfractions, providing the opportunity to develop novel HDL-targeted therapies for the prevention of atherosclerosis.
Abstract 371: An Intact Autophagy-lysosomal System is Required for the Clearance of p62-enriched Inclusion Bodies in Macrophages and has Implications for Atherosclerosis
Authors: Sergin Ismail , Emanuel Roy , Razani Babak
Published: March 2022

Autophagy is an essential catabolic cellular mechanism that involves degradation of dysfunctional proteins and organelles through the lysosomal machinery. Recent studies suggest that autophagy- deficiency induces atherosclerotic plaque formation in mouse models. Although the exact mechanisms are unknown, hyperactivation of the inflammasome, enhanced apoptosis/oxidative stress, and disrupted lipid efflux have been proposed. Herein, we describe an alternative mechanism involving the clearance of protein aggregates. It is known that autophagy-deficient macrophages have significantly increased levels of the chaperone protein p62. Since p62 binds polyubiquitinated proteins and facilitates their turnover via autophagy, disruptions in this process might be pathogenic in atherosclerosis. We first tested the effects of atherogenic lipid loading in peritoneal macrophages. Incubation with either cholesterol crystals or oxidized-LDL dramatically increased p62 protein levels with little effect on transcription, suggesting defects in the clearance of p62 protein. This coincided with the accumulation of polyubiquitinated proteins colocalizing with p62 present either as cytoplasmic inclusions or partially sequestered by lysosomes. Such accumulation was intensified in autophagy-null (ATG5-/-) macrophages, where massive cytoplasmic ubiquitin-positive p62 aggregates form. Our
in vitro
observations are recapitulated
in vivo
; aortas from atherosclerotic (ApoE-/-) mice have progressive and marked elevations in p62 and polyubiquitinated protein levels that predominantly colocalize with plaque macrophages, a process that is further exacerbated in the autophagy-deficient setting. The homeostasis of cytoplasmic inclusions is dependent on the presence of p62 as lipid loaded p62-null macrophages showed polyubiquitinated protein accumulation present in a diffuse and disrupted cytoplasmic pattern. This appears to have functional consequences with p62-null macrophages displaying increased secretion of IL-1β likely through inflammasome activation. Taken together these data suggest that p62-positive inclusion body formation is a marker of atherosclerotic progression with potential pathophysiological consequences.
Abstract 372: CD36 Signaling Regulates Autophagy to Limit Macrophage Cholesterol Accumulation
Authors: Ouimet Mireille , Mak Esther , Franklin Vivian , Marcel Yves L , Moore Kathryn J , Chen Yong-Xiang
Published: March 2022

Autophagy regulates cholesterol mobilization from lipid droplets, a rate-limiting step in macrophage reverse cholesterol transport. Here we investigated the molecular mechanisms of autophagy activation in macrophage foam cells and atherosclerotic plaque. Immunohistochemical staining of human atherosclerotic lesions for LC3 revealed elevated levels of this autophagy marker, particularly in macrophages located in the rupture-prone ‘shoulder region’ of the lesions. This suggests a ‘frustrated autophagy’ in these macrophages, which play important roles in the transition of plaques to a vulnerable state. Notably, we find that macrophages faced with prolonged lipid challenge in vitro exhibit a progressive defect in autophagic flux over time. Pharmacologic stimulation of autophagy in lipid-loaded macrophages enhances cholesterol efflux, suggesting that increasing arterial macrophage autophagy may be anti-atherogenic. However, the molecular mechanisms by which autophagy is stimulated in macrophage foam cells are unclear. Autophagy is rapidly activated in macrophages exposed to oxLDL. Given the role of scavenger receptors in the internalization and signaling response to oxLDL, we investigated whether autophagy activation in foam cells requires CD36. Using Cd36-/- macrophages, we find that oxLDL-induced autophagy is severely impaired compared to WT. Interestingly, preventing dynamin-dependent oxLDL uptake by CD36 does not alter oxLDL-induced autophagy, suggesting that CD36-signaling is sufficient to trigger autophagic activation. Previous studies have shown that CD36 mediates autonomous signaling via Src and MAP kinases, or pairs with Toll-like receptors, including TLR4, to induce the signaling response to oxLDL. Using macrophages from knockout mice, we show that oxLDL-triggered autophagy is intact in the absence of TLR4 or its adaptor MyD88. However, selective inhibition of Src kinases or the MAP kinases Erk and Jnk abolishes induction of LC3 in response to oxLDL. Accordingly, we observe a marked reduction in autophagy levels in plaque macrophages from Apoe-/-Cd36-/- mice on western diet. Together, our data indicate a role for CD36 signaling in limiting cholesterol accumulation in plaque macrophages via induction of autophagy.
Abstract 377: Development of a Cell-Based Assay for In Vitro Assessment of Ultrasound Enhanced Bevacizumab Release from Echogenic Liposomes for Delivery into Atherosclerotic Adventitia
Authors: Klegerman Melvin E , Naji Ali K , Golunski Eva , Peng Tao , McPherson David D
Published: March 2022

Introduction.

Vascular endothelial growth factor (VEGF) is an important mediator of angiogenesis, not only in tumor growth, but also in vulnerable atheroma progression. Recent research has indicated that bevacizumab, an anti-cancer monoclonal antibody targeting VEGF, can inhibit atheroma progression. We have developed bevacizumab-loaded echogenic liposomes (BEV-ELIP) as an ultrasound-triggered intrinsically targeted controlled release vehicle for treatment of developing atheroma.

Hypothesis.

PMA-treated human umbilical vein endothelial cell (HUVEC) cultures can serve as a means of optimizing BEV-ELIP inhibition.

Methods.

Bevacizumab (1 μg/ml) was added to 18-hour HUVEC cultures in 96-well plates, followed by 10 nM phorbol 12-myristate 13-acetate (PMA) 4 hours later and incubated for 24 hours. Controls consisted of no PMA, no BEV; no PMA + BEV; and no BEV + PMA. Cells were enumerated using a hemacytometer and tetrazolium dye MTT.

Results.

PMA enhanced cell proliferation by 21-50% relative to control cultures, but bevacizumab treatment of PMA cultures inhibited cell proliferation by 30-55%, to levels lower than no PMA + BEV controls (Fig. 1). Cell proliferative effects were paralleled by both cell-associated and secreted VEGF levels (determined by western blot and ELISA, respectively).

Conclusions.

We have developed an in vitro cell-based assay for testing BEV-ELIP efficacy with and without ultrasound pretreatment, involving addition of a BEV-containing sample before PMA and determining the inhibition of cell proliferation. This allows us to optimize our therapeutic prior to testing in an in vivo developing atheroma model.
Abstract 379: Potential Protective Effects of Semaphorin3A on Macrophage Function
Authors: Heidari Maryam , Ebrahimian Talin , Lemarie Catherine A , Simon David , Rivas Daniel , Vali Hojatollah , Mandato Craig A , Lehoux Stephanie
Published: March 2022

Semaphorin3A (SEMA3A) is a secreted member of the Semaphorin family, first found to play a role in axon guidance. Recently many studies revealed immune functions for this protein. However, how SEMA3A may regulate monocyte and macrophage functions associated with atherosclerosis is unknown.

An early step in plaque formation is the adhesion of monocytes on activated endothelial cells expressing adhesion molecules. However, we found that 25-100 ng/ml SEMA 3A did not affect the adhesion of monocyes on ICAM-1- or VCAM-1-coated plates. Similarly, stimulation of mouse carotid arteries with SEMA3A in an ex-vivo perfusion system, either administered alone or in conjunction with TNF-α, did not increase adhesiveness of monocytes injected in the intraluminal compartment. Furthermore SEMA3A did not stimulate monocyte migration in a boyden chamber assay. However SEMA3A had significant effects on macrophages, the most abundant cells in the plaque. Inefficient apoptotic cell phagocytosis and macrophage retention in the artery wall are hallmarks of atherosclerosis progression. We found that SEMA3A increased the migration of M2 “wound healing” macrophages more than 4-fold (p<0.05), but had no such effect on M1 “inflammatory” macrophages. Increased M2 migration was associated with phosphorylation of FAK Y397, and could be inhibited by RGD peptide that prevents integrin-matrix interaction and by a blocking antibody targeting the SEMA3A receptor Neuropilin-1. SEMA3A (100ng/ml) also significantly increased phagocytosis of apoptotic cells by M2 macrophages (1.8-fold, p<0.01) but not M1 cells. Interestingly, Sema3A did not affect lipoprotein uptake by macrophages, which characterizes foam cell formation. Finally in an in vivo model of acute inflammation, SEMA3A plasmid pretreatment reduced redness, swelling, and macrophage infiltration associated with LPS injection.In summary, our data suggest that SEMA3A is unlikely to participate in atherosclerotic plaque formation. On the contrary, effects of SEMA3A on macrophage function in vitro and in vivo indicate a potential protective effect in the inflammatory setting.
Abstract 385: Conversion of Established Murine Inducible T Regulatory Cells to Foxp3+IFN?+ T Cells in Atherosclerosis Occurs in an IL-12 Dependent Manner
Authors: Butcher Matthew J , McGary Christopher M , Galkina Elena V
Published: March 2022

In inflammation, T cell subsets are known to play diverse roles and recent evidence suggests that T helper cells display limited plasticity under specific stimuli. Evidence indicates that a decrease in the number of circulating Tregs corresponds with atherogenesis, and the existence of a population of Foxp3+ T regulatory cells that co-expresses inflammatory cytokines such as Interleukin-17A (IL-17A) and Interferon γ (IFNγ) has been demonstrated in models of inflammation. However, the cellular precursors and mechanisms underlying the generation of Foxp3+IL-17A+ and Foxp3+IFNγ+ T cells are unclear. Thus, we sought to study the fate of T regulatory cells during atherogenesis within 12 week western diet (WD)-fed Apolipoprotein E-deficient (Apoe-/-) mice. Apoe-/- mice have a population of Foxp3+IFNγ+ T regulatory cells within the aorta, spleen, peripheral lymph nodes (PLNs), peri-aortic LNs (PALNs), and mesenteric LNs (MLNs). To determine whether Foxp3+IFNγ+ Tregs arise from de novo differentiating naïve T cells or established Tregs, we performed a series of fate tracking experiments with co-adoptively transferred naïve T cells and inducible T regs in Apoe-/- mice. Both adoptively transferred T regs and naïve T cells were present in the blood and emigrated to the aorta, spleen, PLNs, MLNs, and PALNs of Apoe-/- mice. While naïve T cells were present in the tissues examined, they failed to appreciably differentiate to effector T cell fates. In contrast, half of the adoptively transferred Treg cells lost the expression of Foxp3 within all of the tissues examined. The remaining T regs maintained Foxp3 expression (40-45% of transferred Tregs) or adopted a Foxp3+IFNγ+ phenotype (5-10%). To assess how established T reg cells may convert to Foxp3+IFNγ+ T cells, in vitro differentiated iTregs were maintained in Treg conditions with or without IL-12 and assessed by flow cytometry. iTregs cultured without IL-12 did not become Foxp3+IFNγ+ cells. However in iTreg cultures supplemented with IL-12, 10% of Foxp3+ Tregs co-expressed IFNγ. Together, these results suggest that the pro-inflammatory conditions during atherogenesis affect the maintenance of established Treg cells, resulting in the loss of Foxp3 expression or the generation of Foxp3+IFNγ+ Tregs.
Abstract 386: Air Pollutants Induce Dysfunctional Prooxidant HDL and Activate 5-lipoxygenase in the Liver
Authors: Yin Fen , Lawal Akeem , Ricks Jerry , Fox Julie R , Larson Tim , Navab Mohamad , Fogelman Alan , Rosenfeld Michael , Araujo Jesus A
Published: March 2022

Background

Exposure to ambient particulate matter is associated with increased cardiovascular ischemic events and enhanced atherosclerosis.

Objective

To test our hypothesis that air pollutants may induce oxidative modifications of plasma lipoproteins resulting in alteration of the anti-oxidant and anti-inflammatory properties of high-density lipoproteins (HDL).

Methods and Results

We exposed ApoE-/- male mice (C57BL/6 background) to diesel exhaust (DE) emissions at 250 μg PM2.5/m3 for two weeks, filtered air (FA) for two weeks or DE for two weeks, followed by FA for one week (DE+FA). HDL anti-oxidant capacity was assessed by a DCF-based cell fluorescence assay that evaluate the ability of HDL to inhibit LDL oxidation. DE and DE+FA mice significantly impaired the HDL anti-oxidant capacity in comparison with FA mice (p<0.001), as judged by almost a doubling in the level of DCF fluorescence and by a promotion rather than inhibition of oxidation. Decreased HDL anti-oxidant properties were accompanied by reduced anti-inflammatory capacity. The HDL anti-oxidant dysfunction correlated with decreased paraoxonase enzymatic activity, increased levels of plasma 8-isoprostanes, 12-HETE, and 13-HODE. DE also led to increased hepatic levels of ALOX5 mRNA and protein, Glutathione Peroxidase 6 mRNA and 5-HETEs, indicating activation of the 5-lipoxygenase pathway in the liver. Moreover, DE led to enhanced lipid peroxidation in the bronchoalveolar lavage fluid as well.

Conclusions

DE emissions induced systemic pro-oxidant effects that led to the development of dysfunctional HDL, which might be one of the mechanisms how air pollution contributes to enhanced atherosclerosis
Abstract 391: Both STAT3 Activation and Cholesterol Efflux Contribute to the Anti-inflammatory Effect of ABCA1/apoa-interaction
Authors: Tang Chongren , Storey Carl , LeBoeuf Renee
Published: March 2022

Macrophages play a central role in atherogenesis through accumulation of lipids and production of inflammatory cytokines. ATP-binding cassette transporter A1 (ABCA1), a cell membrane protein that is essential for HDL synthesis and reverse cholesterol transport, appears to protect against cardiovascular diseases (CVD) by several mechanisms that include removal of cholesterol from arterial wall macrophages and suppression of inflammation. We have shown that in addition to its cholesterol export activity, ABCA1 functions as an anti-inflammatory signaling receptor through activation of the STAT3 pathway independent of its cholesterol export activity. Several studies have suggested that altering cholesterol content of macrophages can affect their inflammatory activities as well. To dissect the role of lipid export and STAT3 activation on the anti-inflammatory effect of apoA-I or its mimetic peptides /ABCA1 interactions, we created an ABCA1 mutant, ABCA1/Y924F-Y1990F with normal lipid export activity but lacking STAT3 activation, and an ABCA1 mutant, ABCA1/W590S, which has normal STAT3 activation capacity but lacking lipid export activity. We transfected ABCA1/WT, ABCA1/Y924F-Y1990F and ABCA1/W590S using lentivirus techniques back into peritoneal macrophages isolated from mice lacking ABCA1. We found that activation of the STAT3 pathway by the apoA-I or its mimetic peptides/ABCA1 interaction without cholesterol efflux is sufficient to significantly decrease inflammatory cytokines expression. Interestedly, increasing cholesterol efflux by the interaction of ABCA1 with apoA-I or its mimetic peptides without STAT3 activation can also decrease the inflammatory response in macrophages. The maximal anti-inflammatory effect of ABCA1/apoA-I interaction appeared only when STAT3 pathway was activated and cholesterol efflux was increased. Taken together, those studies suggest that both STAT3 activation and cholesterol efflux contribute to the anti-inflammatory efflux of ABCA1/apoA-I interaction by in a synergistic manner. Identifying apoA-I mimetic peptides and small molecules to activate both cholesterol efflux and STAT3 may be a new strategy in the development of therapeutic agents that target ABCA1 pathways for treating CVD.
Abstract 398: Loss of Both Phospholipid and Triglyceride Transfer Activities of Microsomal Triglyceride Transfer Protein in Abetalipoproteinemia
Authors: Khatun Irani , Walsh Meghan , Hussain M. Mahmood
Published: March 2022

Mutations in microsomal triglyceride transfer protein (MTP) cause abetalipoproteinemia (ABL) characterized by the absence of plasma Apolipoprotein B (ApoB)-containing lipoproteins. In this study, we characterized the effects of various MTP missense mutations found in ABL patients with respect to their expression, subcellular location, and interaction with Protein Disulfide Isomerase (PDI). In addition, we characterized functional properties by analyzing phospholipid and triglyceride transfer activities and studied their ability to support apoB secretion. All the mutants co-localized with calnexin and interacted with PDI. We found that R540H and N780Y, that are known to be deficient in triglyceride transfer activity, also lacked phospholipid transfer activity. Novel mutants S590I and G746E did not transfer triglycerides and phospholipids, and did not assist in apoB secretion. In contrast, D384A displayed both triglyceride and phospholipid transfer activities and supported apoB secretion. These studies point out that ABL is associated with the absence of both triglyceride and phospholipid transfer activities in MTP.
Abstract 401: Relative Population Percentiles of LDL Cholesterol, Non-HDL Cholesterol and Total Cholesterol to HDL Cholesterol Ratio: The Very Large Database of Lipids Study (VLDL-2)
Authors: Elshazly Mohamed B , Martin Seth S , Joshi Parag H , Blaha Michael J , Kulkarni Krishnaji R , Jones Steven R
Published: March 2022

Background

Non-HDL cholesterol (non-HDL-C) and the total cholesterol to HDL cholesterol ratio (TC/HDL-C) are alternatives to LDL cholesterol (LDL-C) in risk assessment and decision-making for lipid-lowering therapy.

Methods

We assigned population percentiles to non-HDL-C, TC/HDL-C and LDL-C in 1,340,614 U.S. adults from the Very Large Database of Lipids (VLDL). The distributions of TC, HDL-C, LDL-C and TG in the VLDL were nearly identical to those in the NHANES 2007-2008 survey. Lipids were measured by ultracentrifugation (Atherotech; Birmingham, AL). LDL-C was estimated by the Friedewald method, excluding patients with TG ≥400 mg/dL (2.3% of sample). We examined the relative values of these lipid markers across equivalent population percentiles.

Results

Spearman correlation (ρ) between non-HDL-C and LDL-C percentiles was high overall, ρ=0.94 but lower in patients with LDL-C <70 mg/dL, ρ=0.51; similar for TC/HDL-C and LDL-C percentiles, ρ=0.56; lower in patients with LDL-C<70 mg/dL ρ=0.13. The equivalent non-HDL-C percentile to LDL-C of 70 mg/dL was 93 mg/dL (guidelines recommend 100 mg/dL); equivalent TC/HDL-C of 2.6, the 15
th
percentile. Similarly, the equivalent non-HDL-C percentile to LDL-C of 100 mg/dL was 125 mg/dL (guidelines recommend 130 mg/dL); equivalent TC/HDL-C of 3.3, the 44th percentile (Table).

Conclusion

Selection of treatment targets based on consistent population percentiles may have implications in risk stratification and selection of guideline target values.
Abstract 405: Therapeutic Potential of Oscimum Tenuiflorum in Prevention and Treatment of Atherosclerosis
Authors: Aluganti Narasimhulu Chandrakala , Vardhan Sangamithra , Parthasarathy Sampath
Published: March 2022

Background

Current experimental and clinical studies show that Oscimum tenuiflorum (commonly known as basil or Tulsi) possess many health benefits. Oscimum is also suggested to be anti-oxidative and anti-inflammatory. Eugenol, an ortho-methoxy phenol, is one of its components. Myeloperoxidase (MPO), an oxidative enzyme, has been implicated in the pathogenesis of atherosclerosis. MPO dependent oxidation of lipoproteins has been implicated in foam cell formation, dysfunctional HDL and abnormalities in reverse cholesterol transport (RCT). In this study we tested whether eugenol and basil extract could inhibit the oxidation of lipoproteins (LDL & HDL) by MPO.

Hypothesis

Whole leaf extract of Ocimum tenuiflorum and eugenol would inhibit the oxidation of LDL and HDL by myeloperoxidase and by copper.

Methods

Basil leaf extract was prepared with water, lyophilized and reconstituted in water. Lipoproteins (LDL & HDL) were isolated from human plasma and their oxidation was performed using copper or MPO in the presence or absence of the basil extract and eugenol. MPO activity was measured using guaiacol and TMB as substrates.

Results

Basil extract and eugenol inhibited MPO enzyme activity against synthetic substrates. In addition, they also inhibited the oxidation of LDL and HDL both by copper and MPO, as measured by conjugated diene formation, TBARS formation, as well as by electrophoretic mobility assays.

Conclusion

In lieu of the reported anti-inflammatory properties of basil leaves, and the importance of oxidative and inflammatory pathways in atherosclerosis, such simple non-pharmacological agents could prove to be valuable adjunt therapeutic agents in the treatment of cardiovascular diseases.
Abstract 407: Use of Micellarized Cholesteryl Ester to Generate Macrophage Foam Cells
Authors: Sengupta Bhaswati , Aluganti Narasimhulu Chandrakala , Parthasarathy Sampath
Published: March 2022

Background

Development of foam cells for Reverse Cholesterol Transport (RCT) studies uses the technique of receptor dependent macrophage loading with radiolabeled acetylated LDL. For various reasons this technique is tedious, less reliable and less reproducible. Our goal is to develop foam cells loaded Cholesteryl Ester (CE) in a receptor independent manner and facile manner.

Methods

We micellarized highly nonpolar Cholesterol or CE in aqueous solution of Lyso phosphatidylcholine (Lyso PtdCho) to generate mixed micelles. RAW 264.7 macrophages and mouse peritoneal macrophages were incubated with Cholesterol or CE (unlabeled and fluorescently labeled-NBD cholesterol)/ Lyso PtdCho mixed micelles. The presence of fluorescent Cholesterol and CE was documented by fluorescence microscopy. Foam cell macrophages were detected by Oil-red O staining.

Results

In the presence of Lyso PtdCho, Cholesterol or CE readily formed mixed micelles, generating a clear solution in a concentration-dependent manner. Incubation of Cholesterol or CE rich micelles with macrophages resulted in the uptake of these micelles as noted by fluorescence and Oil-red O staining.

Conclusions

Foam cells developed by this technique could be used for RCT studies. This technique can be manipulated to enrich cells in vitro with various non-polar molecules and could be used to develop different cell based assays.
Abstract 408: Macrophage-mediated Low Density Lipoprotein Modification Reduces Foam Cell Formation
Authors: Meyer Jason M , Ji Ailing , Cai Lei , van der Westhuyzen Deneys R
Published: March 2022

Macrophage metabolism of LDL-derived cholesterol is thought to be a key process regulating the composition and progression of atherosclerotic lesions, including the generation of cholesterol-loaded macrophage foam cells and the lipid-rich necrotic core. Many mechanisms contribute to macrophage endocytosis of LDL, and we recently reported that selective cholesteryl ester (CE) uptake contributes to LDL-induced macrophage cholesterol accumulation. Selective uptake is expected to result in the net removal of cholesteryl ester from LDL; however, the effect of this and other LDL modifications that may occur during incubation with macrophages is not understood. In the current investigation, we pre-incubated LDL for 1-2 days with macrophages in order to promote macrophage-mediated LDL modification. Surprisingly, when macrophage-conditioned LDL was transferred to fresh macrophages, much less cholesterol accumulation resulted compared to cells incubated with unconditioned LDL, as indicated by cholesterol quantification and by neutral lipid staining with Oil Red-O. This difference was not accounted for by changes in uptake of the modified 125I-LDLs, but instead was associated with a large decrease in selective [3H]CE uptake. In contrast, uptake of the non-hydrolyzable CE analog [3H]cholesteryl ether was not reduced, suggesting that reduced uptake in macrophage-conditioned LDL may be due to changes in the hydrolysis of LDL-derived CE or its subsequent metabolism. Cell-mediated LDL oxidation was considered as a possible mechanism for changes in selective CE uptake. However, agarose gel electrophoretic mobility of LDL and analysis of LDL cholesteryl esters indicated that reduced selective uptake and foam cell formation did not depend on extensive oxidative modifications of LDL. These results indicate the importance of selective CE and suggest that macrophage-mediated LDL modification may reduce foam cell formation in some circumstances.