bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 596: Androgen Deprivation Therapy--Induced Weight Gain and Atherosclerosis in Prostate Cancer: Differences Between Orchiectomy, GnRH Agonist and Antagonist
Authors: Hopmans Sarah N , Duivenvoorden Wilhelmina C , Werstuck Geoff , Pinthus Jehonathan
Published: March 2022

Cardiovascular disease (CVD) is one of the most common causes of mortality in prostate cancer patients. In prostate cancer treatment androgen deprivation therapy (ADT) is associated with weight gain and development of the metabolic syndrome. However, different modes of ADT can achieve castration. A
post-hoc
analysis of phase III trials suggests that GnRH antagonists are associated with less cardiac events in men with pre-existing CVD during the first year of ADT. We investigated the effects of bilateral orchiectomy, GnRH agonists and GnRH antagonists on the development of CVD in a mouse model. We hypothesize that GnRH antagonists will associate with reduced adiposity and development of atherosclerosis compared to GnRH analogues and orchiectomy.

We used LDL receptor knockout mice (n=12/group) as models for CVD to investigate and compare the effects of orchiectomy, sham surgery, sham surgery plus GnRH antagonist (degarelix) and sham surgery plus GnRH agonist (leuprolide). Longitudinal weight gain (4 months), visceral fat accumulation (CT measurements), fasting blood glucose, glucose tolerance, serum triglycerides, and testosterone levels were studied along with characteristics of aortic atherosclerotic plaques.

Leuprolide-treated mice gained significantly more weight and visceral fat compared to mice treated with degarelix. Significantly lower levels of serum triglycerides and better response to glucose loading were recorded in mice treated with degarelix. The atherosclerotic plaque area in the aortic sinus in leuprolide-treated and orchiectomized mice was significantly larger than in control mice, but not significantly different from control in degarelix-treated mice. The necrotic plaque area with degarelix was significantly smaller compared to leuprolide-treated and orchiectomized mice.

In a preclinical mouse model, the use of GnRH antagonists attenuates weight gain and development of atherosclerosis.
Abstract 601: Nicotinic Acid Prevents and Restores Steatohepatitis Together with Amelioration of Postprandial Dyslipidemia.
Authors: Hara Masumi , Kurano Makoto , Tsuneyama Koichi , Kikuchi Kentaro , Takai Atsuko , Matsushima Teruhiko , Tsukamoto Kazuhisa
Published: March 2022

Background and aims:
ICR mouse neonatally administered with monosodium glutamate (MSG) shows obesity, insulin resistance, and is a murine model of steatohepatitis. We analysed the effect of nicotinic acid (niacin), an antilipidemic drug and also a potent lipolysis inhibitor.

Methods:
Neonatal ICR mice were subcutaneously injected with 2 mg/g of MSG or saline at 5 consecutive days after birth and fed a normal chow. Niacin was administered by dietary supplementation (0.5% w/w) since 5 week-old. After 12 weeks, mice were sacrificed and tissue samples were collected. Niacin was also administered to 12 week-old mice already having steatosis for 12 weeks.

Results:
After 12 weeks of treatment of niacin, MSG-treated mice showed less weight of body (21.5%, p < 0.001) and liver (52.5%, p < 0.001). Histopathological analyses showed markedly prevention of steatohepatitis. NAFLD activity scores (NAS) of niacin and vehicle groups were 0.0 +/- 0.0, 5.2 +/- 0.4, respectively (p < 0.01). Niacin also dramatically ameliorated accomplished steatohepatitis. Niacin treatment ameliorated dyslipidemia; atherogenic LDL fraction was disappeared in fasting plasma, and elevations of postprandial NEFA and triglycerides were suppressed. Hepatic gene expression of lipogenic genes was not decreased, while expression of Fsp27 was slightly (25%, p < 0.05) decreased. On the other hand, niacin-treated group showed postprandial hyperglycemia. Although there were no evidence of insulin resistance by insulin tolerance test and hepatic Akt phosphorylation, postprandial insulin response was impaired by niacin treatment.

Conclusion:
Niacin prevented and ameliorated steatohepatitis together with amelioration of postprandial dyslipidemia at least partially due to inhibition of NEFA influx into the liver, although niacin impaired postprandial insulin response.
Abstract 602: Depot-Specific Angiogenic Pathways in Human Adipose Tissue: Changes With Type 2 Diabetes
Authors: Haynes Bronson A , Hatcher Margaret A , Junkins David , Hara Manami , Dobrian Anca D
Published: March 2022

Emerging evidence suggests that impaired adipose tissue (AT) angiogenesis leads to metabolic dysfunction. Differences in angiogenesis between the omental (OM) and subcutaneous (SC) depots were previously reported. The peri-pancreatic fat (PF) mass correlates with BMI in humans and may be a significant contributor to local inflammation in islets in type2 diabetes (T2D); however this depot was not yet characterized in humans. The aim of this project was to determine the depot-specific angiogenesis in human obesity and T2D.

OM, SC and PF fat were collected from obese non-diabetic (ND) subjects (BMI>33). Capillary density assessed as CD31 immuno-positivity was 1.25-fold higher in the SC vs. OM AT (p=0.0033). Angiogenic sprouting from matrigel embedded AT explants showed a 1.3-fold increase in the number of branches in SC compared to OM (p<0.05). Unexpectedly, the PF explants showed a complete absence of sprouting in similar conditions. To identify changes in angiogenic genes, we used RT
2
Profiler
TM
Human Angiogenesis PCR Pathway-Focused Array. In OM AT the highest pro-angiogenic expressors were FGF-1 and CXCL6. PF fat showed a 13-fold higher expression of thrombospondin-1 (TSP1) as well as a 13-fold down-regulation of VEGF-C compared to the SC. This result suggests that TSP1 may be key for the angiogenic suppression in PF.

We also examined functional angiogenesis and gene expression in OM and SC from T2D subjects (BMI>33, HbA1c>6.5%). In T2D, capillary density was decreased for SC (p=0.0065) and OM (p=0.0896) AT vs ND. Capillary sprouting in OM and SC explants was significantly reduced by 40% in T2D samples compared to ND. Gene expression of CD34
+
CD31
+
endothelial cells (EC) isolated from T2D SC showed a 4-17 fold down-regulation of CXCL6, IL-6, and IL-8 as well as HIF-1α compared to OM suggesting reduced hypoxia and inflammation. Also, TSP1 was 4-fold increased in EC from OM of T2D subjects. In conclusion, T2D OM and SC fat displayed reduced functional angiogenesis in vitro compared to ND. Gene expression analysis suggests depot-specific pathways regulating angiogenesis in obesity and T2D. TSP1 emerges as a key inhibitor of angiogenesis in human AT and the absence of functional angiogenesis in PF AT is of significance as it could contribute to peri-islet inflammation.
Abstract 609: Pharmacological Inhibition of NLRP3 Inflammasome by Parthenolide Improves Obesity-Induced Insulin Resistance in Diet-Induced Obese Mice
Authors: Nandave Mukesh , Chinta Pavan K , Tambe Suhas , Umrani Dhananjay
Published: March 2022

Many landmark studies have reported the Nod-like receptor proteins containing Pyrin domain (NLRP3) inflammasome activation in metabolic diseases that include obesity, atherosclerosis and type 2 diabetes. Therefore, the present study was aimed: (i) to determine the role of NLRP3 in inflammation and insulin resistance in high fat diet-induced obesity (DIO) model of mice, and (ii) to determine whether parthenolide, a NLRP3 inhibitor, is able to protect mice against inflammation and insulin resistance in high fat DIO model.

METHODS:
Lipopolysaccharide (1 ng/ml) primed mouse intraperitoneal macrophages were treated with Parthenolide (0.1 to 30 μM) to evaluate its effect on TNF-α and IL-1β. Parthenolide and Pioglitazone were administered to DIO mice (fed 60% high fat diet) at 5 and 30 mg/kg QD, PO, respectively for 60 days to evaluate their effect on insulin resistance.

RESULTS:
Parthenolide (5 mg/kg) markedly attenuated inflammatory cytokines as evidenced by significant and dose dependant inhibition of both TNF-α and IL-1β in LPS primed macrophages. Treatment also lowered the fed blood glucose from day 14 to the entire course of study supporting the hypothesis that attenuating inflammation would be a possible intervention to insulin resistance. Parthenolide also improved peripheral insulin resistance, as demonstrated by insulin tolerance test and a significant improvement in the glucose intolerance as demonstrated by the oral glucose tolerance test. Moreover, treatment lowered plasma insulin levels indicating a trend in the improvement in insulin sensitivity.

CONCLUSIONS:
Collectively, obtained results support our hypothesis that Parthenolide, an inflammasome inhibitor, could be a useful agent in antagonizing obesity-induced insulin resistance
Abstract 61: T Cell--Specific Deficiency of ABCG1 Protects Against Atherosclerosis
Authors: Cheng Hsin-Yuan , Wu Runpei , Nowyhed Heba , Hedrick Catherine C
Published: March 2022

ATP-binding cassette transporter G1 (ABCG1) promotes cholesterol efflux from peripheral cells to HDL particles, which transport cholesterol to liver for processing for excretion. ABCG1, and not ABCA1, was found to be critical for the proliferation of T lymphocytes. Despite this, to date, very little is known about the role of ABCG1 in T cells during atherogenesis. In this study, we aim to understand how ABCG1 regulates T cell function and how absence of ABCG1 selectively in T cells impacts atherosclerosis. We found that, on a high cholesterol diet, mice with T cell-specific ABCG1 deficiency on the LDLR
-/-
background (LCK-Cre
+
/ABCG1
fl/fl
/LDLR
-/-
) developed 40% less atherosclerotic lesions than their littermate controls (LCK-Cre
-
/ABCG1
fl/fl
/LDLR
-/-
) (P<0.0001). Furthermore, we found that the percentage of CD4
+
CD25
+
Foxp3
+
regulatory T cells was increased in the LCK-Cre
+
/ABCG1
fl/fl
/LDLR
-/-
mice compared to the littermate controls (P<0.01). Since Tregs are considered anti-atherogenic, we hypothesize that the deficiency of ABCG1 in T cells confers changes in Tregs, which exerts our observed atheroprotective effect. We found that Tregs are hyperproliferative in the LCK-Cre
+
/ABCG1
fl/fl
/LDLR
-/-
mice. We have also found increased conversion of naïve CD4
+
T cells into Tregs in the LCK-Cre
+
/ABCG1
fl/fl
/LDLR
-/-
mice both
in vivo
and
in vitro
. Furthermore, the Treg proliferation and differentiation can both be enhanced by the addition of excess cholesterol
in vitro
. To determine if deficiency of ABCG1 in Treg cells alone is sufficient to drive their induction, we examined the T cell compartment in wild-type and Treg-specific ABCG1 deletion mice (Foxp3-Cre
+
/ABCG1
fl/fl
). The percentage of Tregs in Foxp3-Cre
+
/ABCG1
fl/fl
mice was significantly higher than that of wild-type controls (P<0.01). In short, we have provided evidence that ABCG1 deficiency in T cells leads to decreased atherosclerosis and induction of Tregs. By tying these two findings together, our study provides novel insights in sterol-mediated regulation of Tregs and its impact on atherosclerosis.
Abstract 618: Novel High-Resolution Mass Spectroscopy Methodologies to Study Anti-inflammatory Nitro Fatty Acids
Authors: Petriello Michael C , Sunkara Manjula , Hennig Bernhard , Morris Andrew
Published: March 2022

It is widely hypothesized that the protective effects of certain polyunsaturated fatty acids involves the enzymatic or non-enzymatic conversion to biologically active metabolites. Accordingly, definitive identification and quantitation of bioactive fatty acid derivatives is essential for both evaluating the beneficial effects of these nutrients and identifying biological markers to monitor the therapeutic efficacy of fatty acid administration. Nitro-fatty acids are a class of fatty acid metabolites that are formed by chemical nitration of the double bonds of unsaturated fatty acids, and in mouse models, nitro-fatty acids decrease oxidative stress and inflammation suggesting that these could be biologically relevant mediators. However, due to inherent limitations of currently used analytical approaches for the detection and quantitation of free and esterified nitro-fatty acids, many questions remain to be elucidated regarding their abundance and clinical efficacy. Thus, we hypothesize that using an AMMP derivatization extraction procedure coupled with the use of HR electrospray ionization tandem mass spectrometry and stable isotope dilution will lead to a more accurate profile of nitro-fatty acids in biological samples. To accomplish this we generated methyl pyridinium derivatives of nitro-fatty acids which ionize strongly in positive mode resulting in increased sensitivity of more than 1000-fold over underivatized fatty acids monitored in negative ionization mode. We next developed methods for analysis of methyl pyridinium derivatives of nitro-fatty acids using an AB Sciex 5600 quadrupole TOF MS. Together, these methods allow us to identify the ions of the parent nitro-fatty acid derivative at high resolution and confirm their identities by other techniques such as mass defect and isotopomer ratio pattern analyses. To validate these methods, plasma was collected from C57BL/6 mice and levels of nitro-oleic acid were successfully determined using TOF product ions of 386.31 and 433.31 as key identifiers. In conclusion, we have developed and validated novel analytical methodologies to study nitro-fatty acids which may aide in advancing research of nitro-fatty acids as a therapeutic agent against inflammation and atherosclerosis.
Abstract 621: Mice Lacking Prostaglandin E Receptor Subtype 4 Resist Diet-Induced Obesity Despite Hypertriglyceridemia
Authors: Cai Yin , Vanhoutte Paul M , Tang Eva H
Published: March 2022

Objectives:
Activation of prostaglandin E receptor subtype 4 (EP4) may suppress inflammation by regulating nuclear factor kappa B (NFκB). Indeed, chronic inflammation usually is accompanied by dyslipidemia. Thus, the present study tested the hypothesis that deletion of EP4 can accelerate the occurrence of metabolic syndrome

Methods:
EP4 wild-type and knockout mice were put on a high-fat diet (HFD) for thirty weeks. Body weight and food consumption were monitored. In addition, energy expenditure and fat composition were determined by indirect calorimetry and Minispec body composition analyzer, respectively. The impact of EP4 deletion on the ability to synthesize hepatic very low density lipoprotein (VLDL)-triglyceride (TG) and intestinal chylomicron (CM)-TG, as well as the ability to clear TG during HFD was examined. Circulating lipoprotein lipase (LPL) activity and tissue LPL activity were determined using a fluorometric assay kit and mRNA expression of lipid metabolism-related genes in adipose tissue was measured by quantitative polymerase chain reaction (Q-PCR).

Results:
After consuming a high-fat diet for thirty weeks, EP4 knockout mice exhibited reduced body weight gain and body fat mass, not attributable to changes in food intake, fat malabsorption and energy expenditure. However, EP4 knockout mice had a higher plasma TG level than wild-type mice. The deletion of EP4 did not influence hepatic VLDL-TG production or intestinal CM-TG synthesis but impaired TG clearance rate. EP4 knockout mice had a decreased circulating LPL activity, reduced LPL activity in epididymal fat and skeletal muscle, suggesting impaired hydrolysis and uptake of triglycerides in these tissues. Moreover, EP4 knockout mice had a reduced expression of CD36 in brown adipose tissue, which may indicate that the uptake of fatty acids is impaired.

Conclusions:
High-fat feeding of EP4 knockout mice resulted in a lean body phenotype, but caused hypertriglyceridemia resulting from impaired TG clearance, attributed to reduced LPL activity and expression of CD36. The results indicate that EP4 plays a critical role in systemic lipid homeostasis.
Abstract 625: Niacin Improves Atherogenic Postprandial Lipemia in Patients at High Cardiovascular Risk Despite Reduced Cholesterol Efflux From Human Macrophages
Authors: El Khoury Petra , Waldmann Elisa , Parhofer Klaus , Le Goff Wilfried , Guerin Maryse
Published: March 2022

Introduction:
The role of niacin has been called into question by recent outcome trials showing no benefit on cardiovascular events. Still, for certain high risk patients not achieving their LDL-C target niacin remains a significant therapeutic option.

Hypothesis:
Although the effect of niacin on fasting lipids is well established, its impact on cholesterol efflux and lipoprotein metabolism during the atherogenic postprandial state remains indeterminate.

Methods:
We evaluated the effect of extended-release niacin with laropiprant (ERN/LRP) on postprandial lipoprotein metabolism in 10 subjects on stable statin therapy. All subjects received 1g/20mg ERN/LRP for 4 weeks and then 2 g/40mg ERN/LRP for 8 weeks. At each experimental period, all patients consumed a standardized test meal (1100kcal). Postprandial hypertriglyceridemia (HTG) and plasma efflux capacity through THP-1 macrophages and cellular models overexpressing ABCA1, ABCG1 and SR-BI were evaluated.

Results:
Compared with baseline, ERN/LRP significantly improved postprandial HTG by reducing the area under the curve AUC for chylomicrons (-50%, p=0.004), VLDL-1 (-43%, p=0.0001) and VLDL-2 (-28%, p=0.01). These latter reductions induce a concomitant decrease in CETP activity during the postprandial state (-11.7%;p=0.03, -12%;p=0.01, -18%;p=0.0003, -13%;p=0.0006, 2h, 4h, 6h and 8h respectively after fat load). Plasma cholesterol efflux capacity from human THP-1 macrophages was reduced throughout the postprandial phase (AUC:-20%, p=0.05) following ERN/LRP therapy as a result of a specific reduction in ABCA1 dependent efflux pathway (AUC:-10%; p<0.05), whereas other efflux pathways such as SRBI and ABCG1 were not changed.

Conclusions:
ERN/LRP treatment reduces physiological triglyceride rich lipoproteins synthesis which indirectly results in a significant reduction in CETP activity. By this mechanism, intravascular remodeling of HDL is altered, thus affecting regeneration of ApoA1/preβ-HDL, the preferential cellular cholesterol acceptors via ABCA1 pathway. We conclude that Niacin therapy is associated with an overall reduction in the early step of RCT representing therefore one potential mechanistic hypothesis for the disappointing results in recent clinical trials
Abstract 633: Deficiency of Macrophage 23-kDa Stress Protein Enhances Atherosclerosis via Cholesterol Ester Hydrolysis Dysfunction
Authors: Oh Goo Taeg , Jeong Se-Jin , Kim Sinai , Park Yong Mi
Published: March 2022

Background:
Atherosclerosis is characterized by accumulation of modified lipoproteins in intima of aorta leads to the infiltration of monocytes/macrophages. In the atherosclerotic plaque, these macrophages uptake modified lipoproteins and become foam cells. Macrophage 23-kD stress protein (MSP23), a stress-inducible protein, was found for the first time in the macrophages. Recent reports suggested that the shear stress induce MSP23 expression in endothelial cells. However, the role of MSP23 in macrophages has not been studied yet. In this study, we aim to elucidate atheroprotective effect of MSP23 in macrophages.

Methods and Results:
We show that MSP23 is highly expressed in macrophages among atherogenesis related cells. Double deficiency of MSP23/ApoE mice accelerated plaque formation with increased macrophage infiltrations. Biochemical analyses revealed that MSP23/ApoE double deficient mice exhibited a significant increase in total cholesterol in serum when compared to ApoE deficient mice. Macrophages from MSP23/ApoE double deficient mice expressed higher levels of scavenger receptors and took up more modified lipoproteins compared with ApoE deficient mice, but they expressed low levels of ATP binding cassette protein family A member 1 and were defective in cholesterol efflux. Furthermore, we found that macrophage CE mass was increased in MSP23 deficient macrophages. In bone marrow transplantation experiments, we confirmed that MSP23 plays pivotal atheorprotecitve role in macrophage lineage cells.

Conclusions:
In this study, we elucidate that MSP23 inhibits atherogenesis by intestinal cholesterol ester hydrolysis and augmenting cholesterol efflux from macrophages resulting in reducing formation of foam cells. Thus, MSP23 appears to be a promising therapeutic target for enhancing cholesterol efflux.
Abstract 635: Apolipoprotein A-I Inhibits Lipopolysaccharide-Induced Inflammation and Activation of Major Stress Signaling Kinases in 3T3-L1 Adipocytes in an ABCA1-Dependent Manner
Authors: Sultana Afroza , Cochran Blake J , Tabet Fatiha , Rye Kerry-Anne
Published: March 2022

Obesity is characterised by adipose tissue inflammation and increased secretion of pro-inflammatory adipokines, such as monocyte chemoattractant protein-1 (MCP-1) and interleukin-6 (IL-6). A recently suggested mechanism linking obesity with its co-morbidities is an increase in stress signalling pathways, including p38 mitogen-activated protein kinase (p38MAPK), c-Jun N-terminal kinase (JNK) and extracellular signal-related kinase (ERK). Apolipoprotein A-I (apoA-I), the main lipoprotein of high density lipoproteins, has potent anti-inflammatory properties. It is also the primary acceptor of cholesterol that is exported from cells expressing the ATP-binding cholesterol transporter A1 (ABCA1). The role of apoA-I and ABCA1 in the inhibition of adipocyte inflammation is unknown.

Aim:
To determine if apoA-I inhibits inflammation and activation of stress signalling pathways in lipopolysaccharide (LPS)-stimulated 3T3-L1 adipocytes.

Methods:
Mature 3T3-L1 adipocytes were pre-incubated for 16 h with or without apoA-I (1 mg/mL), then stimulated for 6 h with LPS (100 ng/mL). MCP-1 and IL-6 secretion and mRNA levels were quantified by ELISA and qPCR, respectively. 3T3-L1 adipocytes were also pre-incubated with apoA-I, then stimulated with LPS for 10, 20, 30 and 60 min prior to quantification of ERK1/2, p38MAPK and JNK1/2 phosphorylation by western blotting. ABCA1 expression was silenced by transfection with ABCA1siRNA (50 nM) for 24 h.

Results:
Pre-incubation of 3T3-L1 cells with apoA-I reduced the LPS-mediated secretion of MCP-1 from 2666±216 to 983±65 pg/mg cell protein, while IL-6 secretion decreased from 120±22 to 55±11 pg/mg cell protein (p<0.05 for both). MCP-1 and IL-6 mRNA levels decreased by 43- and 5-fold, respectively, (p<0.05 for both). These effects of apoA-I were attenuated in 3T3-L1 cells in which ABCA1 expression was silenced. LPS induced phosphorylation of ERK1/2, p38MAPK and JNK1/2 in 3T3-L1 cells. Pre-treatment of the LPS-stimulated cells with apoA-I inhibited phosphorylation of ERK1/2, p38MAPK and JNK1/2 by 66.2±4%, 58.8±10.7% and 33.2±14% respectively (p<0.05 for all).

Conclusion:
ApoA-I exerts anti-inflammatory effects and inhibits activation of MAP kinases in 3T3-L1 adipocytes in an ABCA1-dependent manner.
Abstract 642: Vascular Endothelial Growth Factor Receptor 2 Blockade in Murine Vein Graft Ameliorates Lesion Growth and Enhances Plaque Stability by Reducing Intraplaque Hemorrhage
Authors: de Vries Margreet R , de Jong Rob C , Peters Erna A , Hamming Jaap F , Goumans Marie-José , Quax Paul H
Published: March 2022

Immature plaque neovessels contribute to atherosclerotic plaque instability and intraplaque hemorrhage by leaking erythrocytes and leukocytes in the plaque. Vascular Endothelial Growth Factor Receptor 2 (VEGFR2), together with the angiopoietin (Ang)-Tie2 system, regulates the maturation of neovessels. We have previously shown that murine vein graft lesions exhibit massive plaque neovascularization and that intraplaque hemorrhage contributes to lesion growth. We hypothesized that blockade of VEGFR2 results in more mature plaque microvessels and less intraplaque hemorrhage.

Donor caval veins were engrafted in carotid arteries of recipient hypercholesterolemic ApoE3*Leiden mice (n=14/group). Mice were treated at day 14, 17, 21 and 25 with VEGFR2 blocking antibodies (DC101) or control IgG antibodies (10 mg/kg). At day 28 mice were sacrificed for histological analysis of the vein grafts.

Morphometric analysis revealed a striking 50% decrease in vein graft segments that expressed intraplaque hemorrhage in the form of leaky vessels in the DC101 treated group. This was accompanied by a significant 25-fold decrease in extravasated erythrocytes. Furthermore, lesions that exhibit intraplaque hemorrhage showed a strong increase in Ang-2, indicative for immature neovessels. VEGFR2 blockade however, did not affect the neovessel density in the lesions (control 52±19 neovessels/section; DC101 63±25 neovessels/section). Interestingly, the vein graft lesion area in the DC101 group was significantly reduced with 32% compared to the control group. Moreover, plaque stability was clearly increased in DC101 treated mice, determined by a 25% reduction in macrophage content, a 50% increase in collagen content and a 120% increase in SMC content.

Blockade of VEGFR2 leads to reduced intraplaque hemorrhage, decreased vein graft lesion area and increased plaque stability. This identifies plaque neovascularization as an attractive target for the treatment of unstable atherosclerotic diseases.
Abstract 648: Visualizing the Genesis of Microcalcifications via Matrix Vesicle Aggregation: Novel Implications for Plaque Vulnerability
Authors: Hutcheson Joshua D , Goettsch Claudia , Ricchiuto Piero , Bertazzo Sergio , Maldonado Natalia M , Aikawa Masanori , Singh Sasha , Aikawa Elena
Published: March 2022

Background:
Microcalcifications in the cap of vulnerable plaques contribute to plaque rupture. The mechanism by which microcalcifications form is poorly understood due to the inability to monitor initiating calcification events in vivo. We hypothesized that aggregation of cellular-derived matrix vesicles (MV) within atheroma lead to the formation of microcalcifications.

Results:
Transmission electron microscopy of ApoE-/- mouse plaques reveals MVs aggregating in regions of disrupted collagen (A). To image aggregation of calcifying MVs, we created a 3D collagen hydrogel system to simulate the atherosclerotic fibrous cap. Conditioned media from calcific human coronary artery smooth muscle cells was added to collagen hydrogels. Confocal microscopy reveals spherical microcalcifications (B, arrows) resembling those previously reported in human plaques. Super-resolution (< 200 nm) microscopy of the collagen hydrogels shows individual MVs aggregating (C, arrows) to form microcalcifications shown by a near-infrared calcium phosphate tracer (D). Scanning electron microscopy combined with x-ray spectroscopy confirms the presence of calcium phosphate mineral within MV aggregates (E, orange color). MV aggregation and calcification size is inversely related to the amount of collagen in our 3D model (p<0.05).

Conclusion:
To our knowledge, we are the first to directly visualize aggregation and calcification of MVs in real time. This approach presents a powerful tool to understand the genesis of microcalcifications from nucleating events. Aggregation of MVs within a thinning fibrous cap leads to the formation of microcalcifications, which further destabilize the cap.
Abstract 655: 4-Phenylbutyrate in Drinking Water Reduced Atherosclerotic Lesion Growth in ApoE-/- Mice by Modifying Monocyte-Macrophage Differentiation
Authors: Lynn Edward G , Lhoták Šárka , Austin Richard C
Published: March 2022

Cardiovascular disease and atherothrombosis account for the majority of deaths worldwide. Plaque rupture, thrombus formation, and vessel occlusion contribute to the acute clinical manifestations of atherothrombosis. Previously we have shown that the unfolded protein response (UPR) has an important role in monocyte-macrophage differentiation. Furthermore, macrophage infiltration, lipid accumulation and foam cell formation are key events in atherogenesis. Endoplasmic reticulum (ER) stress-dependent apoptosis also contributes to atherosclerotic lesion growth and plaque necrosis. The aim of the current study was to examine whether 4-phenylbutyrate (4-PBA), a small chemical chaperone that alleviates ER stress, confers protection against atherosclerosis when provided via drinking water. To investigate the ability of 4-PBA to affect atherosclerotic lesion growth, at 12 weeks of age female ApoE-/- mice on chow diet were provided with 4-PBA (1 - 2.5 g/kg/d) in their drinking water for five weeks, ad libitum. Following treatment, mice were sacrificed, their tissues collected and atherosclerotic lesions examined by immunohistochemistry. Early atherosclerotic lesion area was significantly reduced in ApoE-/- mice provided 4-PBA-treated water from 12 - 17 weeks of age, relative to controls (0.6 x105 ± 0.1 μm2 versus 1.1 x105 ± 0.1 μm2, respectively, P < 0.05). We hypothesized that 4-PBA drinking water decreased lesion size by a mechanism involving reduced ER stress and monocyte-macrophage differentiation. In support of this mechanism, preliminary data indicates 4-PBA inhibited monocyte-macrophage differentiation, as measured by mRNA expression of the macrophage marker MSR-1 (0.5 ± 0.2 fold-change vs. control) and cell adherence (37 ± 5% vs. control) in PMA-treated THP-1 cells. In summary, these findings provide evidence that 4-PBA-treated drinking water reduced atherosclerotic lesion size in a mouse model of atherosclerosis. We hypothesize that 4-PBA acts in part through its ability to reduce ER stress and modulate monocyte/macrophage differentiation and function. Additional studies are in progress to further examine the mechanisms by which 4-PBA affects atherogenesis.
Abstract 659: PKR-Like Endoplasmic Reticulum Kinase and Glycogen Synthase Kinase-3α/ß Signaling Regulates Endoplasmic Reticulum Stress-Induced Foam Cell Formation
Authors: McAlpine Cameron , Werstuck Geoff
Published: March 2022

Background:
Evidence suggests a causative role for endoplasmic reticulum (ER) stress in the development of atherosclerosis. The molecular mechanisms by which conditions of ER stress promote pro-atherogenic processes are not understood. We have found that ER stress-inducing agents can activate glycogen synthase kinase (GSK)-3α/β, a protein involved in many metabolic pathways. The objective of this study is to investigate the role of GSK3α/β in pro-atherogenic ER stress signaling.

Methods and Results:
Thp1-derived macrophages were treated with the ER stress-inducing agents, glucosamine, thapsigargin or palmitate, in the presence or absence of the GSK3α/β inhibitor CT99021. GSK3α/β inhibition did not affect the adaptive unfolded protein response (UPR), but did block ER stress-induced lipid accumulation as well as the up regulation of genes associated with lipid biosynthesis and uptake. Using small molecule inhibitors of specific UPR pathways, we found that PERK, but not IRE1 or ATF6, is required for the activation of GSK3α/β by ER stress. GSK3α/β inhibition attenuated ER stress-induced expression of distal components of the PERK pathway, including CHOP and ATF4. Atherosclerotic plaques from ApoE-/- mice, fed a diet supplemented with the GSK3α/β inhibitor valproate, had reduced levels of CHOP within the macrophage foam cells. In primary mouse macrophages PERK inhibition blocked ER stress induced lipid accumulation whereas the overexpression of constitutively active S9A-GSK3β promoted foam cell formation and CHOP expression, even in cells treated with a PERK inhibitor.

Conclusions:
Pharmacological inhibition of GSK3α/β attenuates ER stress-induced lipid accumulation and macrophage foam cell formation. These findings indicate that GSK3α/β is an important factor in the ER stress-PERK signaling pathway and may play a central role in the pro-atherogenic dysregulation of lipid metabolism.
Abstract 669: SR-BI in Bone Marrow--Derived Cells Protects Mice From Diet-Induced Coronary Artery Atherosclerosis and Myocardial Infarction
Authors: Pei Ying , Chen Xing , Aboutouk Dina , Fuller Mark T , Dadoo Omid , Yu Pei , White Elizabeth J , Igdoura Suleiman , Trigatti Bernardo L
Published: March 2022

Background:
SR-BI deficient, apolipoprotein E hypomorphic mice develop diet induced occlusive coronary artery atherosclerosis, myocardial infarction and early death.

Methods:
To test the role of SR-BI in bone marrow derived cells, SR-BI-null; apoE- hypomorphic mice were transplanted with SR-BI
+/+
apoE-hypomorphic, or control, autologous SR-BI-null; apoE-hypomorphic bone marrow. Four weeks later, mice were fed a high-fat, high-cholesterol, cholate-containing diet to induce coronary artery atherosclerosis.

Results:
Mice transplanted with autologous bone marrow developed extensive aortic atherosclerosis and severe occlusive coronary artery atherosclerosis after 4 weeks of feeding. This was accompanied by myocardial fibrosis and increased heart weights. In contrast, restoration of SR-BI expression in bone marrow derived-cells reduced diet induced aortic and coronary artery atherosclerosis, myocardial fibrosis and the increase in heart weights in SR-BI-null; apoE-hypomorphic mice. Restoration of SR-BI in bone marrow derived cells did not, however, affect steady state lipoprotein cholesterol levels, but reduced plasma levels of IL-6. Monocytes from SR-BI-null mice exhibited a greater capacity to bind to VCAM-1 and ICAM-1 than those from SR-BI
+/+
mice. Furthermore, restoration of SR-BI expression in bone marrow derived cells attenuated monocyte recruitment into atherosclerotic plaques in mice fed high fat, high cholesterol cholate containing diet.

Conclusion:
These data demonstrate directly that SR-BI in bone marrow-derived cells protects against both aortic and coronary artery atherosclerosis.
Abstract 671: Beyond Cholesterol Efflux: Defining HDL Function in Atherosclerosis and the Requirement for Procollagen C-Endopeptidase Enhancer Protein 2
Authors: Pollard Ricquita , Blesso Christopher N , Zabalawi Manal , Fulp Brian , Lyons Erica W , Francone Omar L , Thomas Michael J , Sorci-Thomas Mary G
Published: March 2022

Recent studies suggest that assessment of high density lipoprotein (HDL) function provides a more reliable metric of athero-protection than plasma concentration alone. One important function attributed to apolipoprotein A-I (apoA-I), the major protein constituent of HDL, is its ability to accept newly effluxed cholesterol from ATP-binding cassette A-I (ABCA1) to form nascent HDL (nHDL). New studies suggest that the protein, procollagen c-endopeptidase enhancer protein 2 (PCOLCE2; PCPE2), is involved in mediating nHDL particle formation. PCPE2 is a 52 kDa glycoprotein sharing 43% amino acid identity with PCOLCE (PCPE1). Both are secreted into the extracellular matrix where they enhance the catalytic cleavage of the C-terminal propeptides of procollagen catalyzed by bone morphogenetic protein 1 (BMP1). The connection between PCPE2 and HDL was discovered after significant correlations were found between single-nucleotide polymorphisms and plasma HDL-cholesterol concentrations in population studies. Consistent with these findings a recent genome-wide siRNA screening study indicated that PCPE2 was a significant modulator of HDL apoA-I secretion from cells, while other studies in mice lacking PCPE2 showed defective ABCA1-mediated cholesterol efflux, despite elevated plasma HDL. Thus, we began the current studies to determine if the increased plasma HDL, in light of defective cholesterol efflux, would be atheroprotective or atherogenic. PCPE2 null mice were crossed with LDL receptor null mice to obtain double knockout, LDLr
-/-
, PCPE2
-/-
mice. Both LDLr
-/-
and LDLr
-/-
, PCPE2
-/-
mice were fed an atherogenic diet for 12 wks after which the lipid deposition in the aortic root was evaluated. As expected, LDLr
-/-
, PCPE2
-/-
mice showed elevated levels of enlarged HDL compared to control mice, while LDL levels were similar between the two genotypes. Despite elevations in plasma HDL, LDLr
-/-
, PCPE2
-/-
mice showed significantly more atherosclerosis compared to LDLr
-/-
mice, similar in extent to that found in mice that lack HDL due to an absence of plasma apoA-I (LDLr
-/-
, apoA-I
-/-
). This outcome suggests that PCPE2 is atheroprotective and likely participates at various levels of HDL metabolism, including HDL production, remodeling and catabolism.
Abstract 674: 15(S)-Hydroxyeicosatetraenoic Acid Induces Foam Cell Formation by Enhancing CD36 Expression via Activation of ROS-Syk-Pyk2-STAT1 Signaling
Authors: Kotla Sivareddy , Singh Nikhlesh K , Rao Gadiparthi N
Published: March 2022

15(S)-Hydroxyeicosatetraenoic acid (15(S)-HETE), the major 15-lipoxygenase 1 (15-LO1) metabolite of arachidonic acid (AA), induces CD36 expression in xanthine oxidase and NADPH oxidase-dependent ROS production and Syk and Pyk2-dependent STAT1 activation. In line with these observations, 15(S)-HETE also induced foam cell formation involving ROS, Syk, Pyk2 and STAT1-mediated CD36 expression. In addition, peritoneal macrophages from high-fat diet-fed ApoE-/- mice exhibited elevated levels of xanthine oxidase and NADPH oxidase activities, ROS production, Syk, Pyk2, and STAT1 phosphorylation and CD36 expression compared to those from ApoE-/-:12/15-LO-/- mice and these events correlated well with increased lipid deposits and macrophage content in the aortic arches resulting in atherosclerotic plaque formation. These findings strongly suggest that 12/15-LO metabolites of AA such as 15(S)-HETE play a crucial role in atherogenesis by enhancing foam cell formation.
Abstract 675: Role of Indoleamine 2,3-dioxygenase in Atherosclerosis Development
Authors: Taleb Soraya , Ponnuswamy Padma , Alain Tedgui , Mallat Ziad
Published: March 2022

Indoleamine 2,3-dioxygenase (Ido), a rate-limiting enzyme that catalizes the degradation of tryptophan along the kynurenine pathway, is seen as a major mediator of immune tolerance. Our purpose is to study the role of Ido in an inflammatory disease which is atherosclerosis. We showed that surprisingly the total Ido deletion in Ldlr-/- mice (Ldlr-/-Ido-/-) or in mice reconstituted with Ido-/- bone marrow showed a significant reduction in atherosclerosis (p<0.05). Then, we examined the inflammatory profile of cells known to express Ido such as macrophages. Interestingly, Ido-/- macrophages showed a significant increase of IL-10 expression compared with controls (p<0.05).To examine the role of IL-10 in Ido-mediated effect, we made double knockout- mice Ido-/-Il-10-/-. Importantly, the resistance of Ido-deficient mice to enhanced atherosclerosis and immune activation is broken in Ido-/-Il-10-/- mice, which then show exaggerated immune responses and develop a very severe colitis. We also showed that kynurenic acid, a kynurenine-derived metabolite, is responsible for reduced Il-10 production through inhibition of Erk1/2 phosphorylation and increased of atherosclerosis development (p<0.05). Our results reveal a previously unsuspected Janus-faced Ido that actively represses Il-10 while promoting other pathways of immune tolerance, thereby introducing a higher degree of complexity in immune regulation.
Abstract 69: Characterization of the Role of Glycerol-3-phosphate Acyltransferase-3 in Intestinal Lipid Absorption
Authors: Khatun Irani , Clark Ronald , Lanba Adhiraj , Vera Nicholas , Erion Derek , Coskran Timothy , Bobrowski Walter , Okerberg Carlin , Goodwin Bryan
Published: March 2022

Glycerol-3-phosphate acyltransferases (GPAT) catalyze the first step in the synthesis of glycerolipids and glycerophospholipids. Microsomal GPAT, the major GPAT activity, is encoded by at least two closely related genes, GPAT3 and GPAT4 (also known as AGPAT9 and 8, respectively). The physiological role of GPAT3 is poorly understood. GPAT3 mRNA was found to be abundantly expressed in small intestine which was confirmed by immunohistochemistry. The GPAT3 protein was highly enriched in enterocytes suggesting a potential role in intestinal lipid absorption. To further investigate this possibility, we generated Gpat3-deficient mice (Gpat3-/-). The Gpat3-/- mice were viable and fertile, and exhibited no obvious metabolic abnormalities on standard laboratory chow. As expected, Gpat3-/- mice had reduced intestinal GPAT activity compared to wild-type animals and when challenged with an oral lipid bolus, Gpat3-/- mice displayed attenuated plasma triglyceride excursion. Histological and biochemical analysis of the small intestine revealed dramatically increased lipid deposition in the intestinal mucosa of Gpat3-/- mice. Long chain acylcarnitine levels were elevated in the jejunum of Gpat3-/- suggesting a compensatory increase in mitochondrial fatty acid oxidation. Lipid flux studies performed in primary cultures of enterocytes derived from wild-type and Gpat3-/- mice reveal a defect in triacylglycerol trafficking suggesting a role for GPAT3 in the post-prandial secretion of lipid in the intestine. Finally, gene expression analysis revealed dysregulation of multiple genes involved in lipid absorption, chylomicron assembly, and secretion. Taken together, our results demonstrate a hitherto unappreciated role for GPAT3 in lipid absorption.
Abstract 70:
KLF14
is a Novel Regulator of Human Metabolism
Authors: Ding Qiurong , Gupta Rajat M , Raghavan Avanthi , Musunuru Kiran
Published: March 2022

Human genetic studies have identified DNA variants near the
KLF14
gene to be strongly associated with HDL cholesterol levels, triglyceride levels, risk of type 2 diabetes mellitus, and risk of coronary artery disease. Furthermore, the same variants are associated with both the expression level of
KLF14
(
cis
eQTL) and the expression levels of ten other genes related to metabolic traits located on different chromosomes (
trans
eQTL) in human adipose tissues, suggesting that
KLF14
may be a master regulator of gene expression in adipose tissue and a key player in human metabolism. In order to evaluate this hypothesis
,
we have rapidly and efficiently generated both
KLF14
knockout human adipocytes (differentiated from human pluripotent stem cells) and
Klf14
knockout mice through the use of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems. We have analyzed the
KLF14
knockout adipocytes and the
Klf14
knockout mice for metabolic phenotypes at several different levels. At an organismic level, we found
Klf14
knockout mice displayed a variety of metabolic phenotypes—increased levels of triglycerides (up 61%), free fatty acids (up 75%), free glycerol (up 43%), and cholesterol levels (up 12%) in the blood. At a tissue level, we found
Klf14
knockout adipose showed a broad activation of genes related to lipid synthesis and downregulation of groups of genes involved in muscle function and development, suggesting a switch between muscle (energy usage) and adipose (energy storage) programs. We also confirmed in
Klf14
knockout adipose tissue significant changes in several of the ten
trans
eQTL genes reported in human adipose tissue. At a cellular level, we found
in vitro
differentiated
Klf14
knockout adipocytes had increased adipogenesis with larger lipid droplets and more triglyceride accumulation (increased >50%) compared to wild-type adipocytes. Thus, using novel genome-editing techniques, we were able to rapidly evaluate
KLF14
function in mouse and human model systems. Taken together, the obtained data establish that
KLF14
is a causal regulator of human metabolism, acting at least in part by modulating adipocyte function. Further studies in other metabolically active tissues such as liver and muscle are underway.
Abstract 73: A Novel Lysosome-Mediated Degradation Mechanism for Hepatic Lipid Droplet Turnover
Authors: Amir Alipour Mohsen , Yao Zemin
Published: March 2022

Dietary supplementation with fish oil, rich in n-3 fatty acids such as EPA (20:5n-3), has been shown in many studies to exert a hypotriglyceridemic effect by reducing both plasma triglycerides (TG) in the form of VLDL and intrahepatic TG that is presented in cytoplasm as lipid droplets. However, cellular or molecular mechanisms by which n-3 fatty acid treatment could reduce hepatic TG remain to be defined. We used a well-characterized cell culture system that possesses the ability to synthesize and secreted TG-rich VLDL1 to determine the effect of EPA treatment on the synthesis, storage, turnover, and secretion of TG. Robust accumulation of lipid droplets occurs when hepatic cells were cultured under lipid-rich conditions, and prolonged exposure of the cells to lipids could lead to impaired TG secretion and steatosis. Treatment of the lipid-laden cells with EPA effectively cleared the storage lipid droplets within a few hours. The enhanced turnover of lipid droplets in EPA-treated cells was not attributable to increased TG secretion, because EPA-treatment also impaired VLDL1 assembly/secretion. The droplet turnover required the activity of lysosomes and could be blocked either by NH4Cl treatment or by silencing LAMP1 or Rab7. Inactivation of Atg5, ATGL, or HSL had no effect on the droplets turnover, indicating that the process is independent of autophagosome formation or cytosolic lipase activities. Live cell imaging of cells treated with EPA showed dynamic “kiss-and-run” events between lysosomes and the droplets, which were accompanied with augmented beta-oxidation. Treatment of cells with nocodazole or silencing the Rab7 effctors FYCO1 and RILP, respectively, completely blocked the EPA-induced droplet turnover, suggesting that the movement of lysosomes was essential for the kiss-and-run process. Moreover, overexpression of Arl8b, a lysosome-associated Arf-like GTPase, markedly accelerated the EPA-induced droplet turnover. These results reveal a novel mechanism responsible for the hypolipidemic effect of n-3 fatty treatment, and suggest a therapeutic potential for n-3 fatty acids in the treatment of hepatosteatosis.
Abstract 74: Thrombospondin 1 Modulates Arterial Thrombosis via von Willebrand Factor in Mice
Authors: Prakash Prem , Gandhi Chintan , Ahmad Ajmal , Chauhan Anil
Published: March 2022

Background and Hypothesis:
Thrombospondin 1 (TSP1) is a large adhesive glycoprotein that is stored in platelet α-granules. Upon platelet activation, TSP1 is released in large amount (up to 30 μg/mL) in circulation. Previous studies have shown smaller plasma von Willebrand factor (VWF) multimer size in Tsp1
-/-
mice compared with wild-type (WT), suggesting that TSP1 protects VWF from excessive proteolysis by ADAMTS13. It remains unclear whether TSP1/VWF axis determines the rate of thrombus growth in injured vessels. We tested the hypothesis that TSP1 modulates arterial thrombosis via VWF.

Model and Methods:
We generated
Vwf
-/-
/
Tsp1
-/-
mice and compared with
Tsp1
-/-
,
Vwf
-/-
and WT mice. Platelet adhesion and susceptibility to thrombosis was assessed in a ferric-chloride injury-induced mesenteric artery thrombosis model. Using high-resolution intravital microscopy, we measured the time to form 1
st
thrombus (>20 μm), thrombus growth kinetics and occlusion time in the injured arterioles.

Results:
We found that the number of fluorescently labeled platelets that adhered transiently to 250μm vessel wall segment within 3-4 min following ferric-chloride injury was similar in
Tsp1
-/-
mice compared with WT mice. The mean time to form first thrombus was significantly increased in
Tsp1
-/-
mice compared with WT (9.98±0.52 vs. 7.12±0.55min, P<0.05). The rate of thrombus growth was decreased in
Tsp1
-/-
mice (
P
<0.05 vs. WT). The time to stable occlusion of the mesenteric artery was modestly but significantly prolonged in
Tsp1
-/-
mice compared with WT mice (15.8±0.59 vs. 13.3±0.63min, P<0.05). Because TSP1 binds to A3 domain of VWF, we investigated TSP1 modulates thrombosis via binding to VWF. Compared to
Vwf
-/-
mice,
Vwf
-/-
/
Tsp1
-/ -
littermates exhibited similar platelet adhesion, time to form first thrombus, and non-occlusive thrombus growth kinetics, suggesting that TSP1 modulates thrombus growth via VWF.

Conclusion:
We provide evidence for the first time that TSP1 does not mediate initial platelet adhesion to injured vessel wall but modulates arterial thrombus growth via a mechanism that requires VWF. These results suggest that TSP1 may participate in amplifying platelet aggregation following injury by protecting VWF from excessive cleavage by ADAMTS13.