bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 628: Efficient Excretion of Xenosterols in the Absence of Abcg5/Abcg8
Authors: Patel Shailendra B
Published: June 2021

Hypothesis:
Xenosterol excretion by the mammalian body is wholly dependent on Abcg5/Abcg8 function.

Methods:
To test our hypothesis, we loaded Abcg8-/- mice with dietary plant sterols until they manifested biochemical (elevated plant sterols) and pathological changes (poor body weight, macrothrombocytopenia). All dietary xenosterols were eliminated from the diet and kinetics of xenosterol loss monitored over the ensuing 16 days, monitoring plasma, bile, and stool losses, as well as whole body sterol determinations.

Results:
After loading Abcg8-/- mice with plant sterols, plasma sitosterol levels in both male and female mice averaged 80mg/dL. Surprisingly, there was rapid loss of xenosterols from blood as well as tissues (liver, whole body), with losses of xenosterols detected in bile and feces. By day 16, stool xenosterols were almost undetectable and reflected the dramatic loss of whole body xenosterols.

Conclusions:
Our data refute our hypothesis. Although loss of sterolin function leads to severe xenosterolemia in both humans and mice, at least in mice, there appears to be a pathway(s) for elimination via the biliary/intestinal route. Characterization of the transporters involved may shed further light on sterol trafficking.
Abstract 632: Modification of HDL by γ-Ketoaldehydes Causes impaired HDL Function in Familial Hypercholesterolemia
Authors: Tao Huan , Yancey Patricia G , Huang Jiansheng , Blakemore John L , Zhang Youmin , Ding Lei , Fazio Sergio , Vickers Kasey C , Oates John A , Roberts L. Jackson , Davies Sean S , Linton MacRae F
Published: June 2021

Background:
Dysfunctional high density lipoprotein (HDL) is linked to the pathogenesis of atherosclerosis and cardiovascular events. Lipid peroxidation products have been reported to enhance atherogenesis and compromise HDL atheroprotective functions. The role of γ-ketoaldehyde modification on HDL function and atherosclerosis was examined in the setting of familial hypercholesterolemia (FH) and by using
Ldlr
-/-
mice as an atherosclerotic model.

Methods and Results:
Sandwich enzyme-linked immunosorbent assay (ELISA) of HDL malondialdehyde (MDA) content revealed that HDL from subects with FH contained 5.7-fold (P<0.01) more MDA compared to control HDL. Analysis by Western blot demonstrated that FH HDL contained 5.9- and 3.6-fold (P<0.01) more MDA-apoAI and isolevuglandins (isoLGs)-apoAI adducts compared to control HDL, respectively. Heterozygous (het)-FH versus control HDL was 42% (P<0.01) less efficient at reducing cholesterol stores in apoE
-/-
foam cells. In the presence of LPS, het-FH HDL stimulated 2.9- and 3.3-fold (P<0.05) more expression of IL-1β and TNF-α in apoE
-/-
cells versus control HDL, suggesting impaired anti-inflammatory function. Importantly, IsoLG modification of apoAI decreased the cholesterol efflux capacity by 71% (P<0.01). HDL from
Ldlr
-/-
mice fed a Western versus a chow diet contained 6.2-fold (P<0.01) more MDA-apoAI and stimulated 57% (P<0.05) and 71% (P<0.01) more expression of inflammatory IL-1β and TNF-α. Administration of the ketoaldehyde scavenger, salicylamine (SAM), versus the nonreactive analogue, 4-SAM, to
Ldlr
-/-
mice consuming a Western diet reduced the HDL MDA-apoAI content by 55.8% (P<0.01). Importantly, 3 months of SAM treatment reduced atherosclerosis of
Ldlr
-/-
female mice fed a Western diet for 16 weeks by 42% (P<0.01) as analyzed by Oil-Red-O staining of proximal aortic sections.

Conclusions:
Our studies suggest that FH HDL function is impaired as a result of increased MDA and isoLG modification and identify ketoaldehyde scavenging as a potential therapeutic means to prevent atherosclerosis development.
Abstract 638: Response Gene to Complement 32 Promotes Atherosclerosis Through Endothelial Cell Dysfunction Induced Vascular Inflammation
Authors: Cui Xiaobing , Luan Junna , Chen Shiyou
Published: June 2021

Atherosclerosis is the major cause of cardiovascular diseases and the primary cause of death worldwide. Our previous studies have demonstrated that response gene to complement 32 (RGC-32) deficiency prevents the mice from high-fat diet-induced obesity and insulin resistance. The present study was conducted to determine the role of RGC-32 in the development of atherosclerosis. We observed that endothelial RGC-32 expression was dramatically induced in human and mouse atherosclerotic lesions. RGC-32 deficiency (RGC-32-/-) significantly attenuated the atherosclerotic lesion formation in arteries of ApoE-/- mice. Mechanistically, RGC-32-/- diminished the expression of vascular cell adhesion molecule (VCAM)-1 and intercellular adhesion molecule (ICAM)-1 in primary cultured mouse endothelial cells induced by tumor necrosis factor (TNF)-α. RGC-32-/- also inhibited TNF-α-induced monocyte-endothelial cell interaction. These data demonstrate that RGC-32 contributes to the development of atherosclerosis by regulating the expression of endothelial adhesive molecules, and thus facilitating macrophage infiltration. Therefore, RGC-32 may be a potential novel drug target in the treatment of atherosclerosis and its related cardiovascular diseases.
Abstract 652: Coronary Angioscopic Observation of Extremely Late Arterial Repair Catch-up after Sirolimus-eluting Stent Implantation
Authors: Ichikawa Minoru , Bando Kazunori , Kijima Yoshiyuki
Published: June 2021

Sirolimus, a bio-suppressive drug, delays coronary arterial repair after implantation of Cypher sirolimus-eluting stents (SES), a representative first generation drug-eluting stent. It remains unclear, however, whether this delayed repair would catch up with bare metal stents (BMS) along extremely long observation. The aim of this study was angioscopic comparison of coronary arterial repair through a decade after implantation of SES and BMS. Enrolled were 31 patients with previous implantation of SES (n = 22) or BMS (n = 9). Duration after stent implantation (DSI) ranged from 3 to 10.5 years in both groups. Thirty-seven SES were implanted into the SES group and 17 BMS into the BMS group. The 54 stented segments were analyzed by quantitative coronary angiography (QCA) and non-obstructive coronary angioscopy (CAS). Under continuous flush of low-molecular-weight dextran, CAS visualized in-stent appearance, i.e. endothelial stent coverage (ESC), presence of yellow plaque (YP), and in-stent mural thrombi (ISMT). ESC was semi-quantified into 4 grades (grade 0, no coverage; grade 1, thin coverage; grade 2, decent coverage; grade 3, full coverage). QCA showed in-stent restenosis neither in SES- nor BMS-implanted segments (% diameter stenosis 14.8 ± 7.9 vs. 16.2 ± 8.2, respectively). Incidence of YP was 27% in SES and 6% in BMS (P < 0.03). Incidence of ISMT was 24% in SES, whereas never in BMS. ESC grade of BMS was either grade 1 (24%) or grade 3 (76%). SES-implanted segments were divided into two groups, i.e. DSI > 8 years (n = 5) and DSI < 8 years (n = 32). In the former (10.5 > DSI > 8 years), ESC grade of SES was either grade 1 (40%) or grade 3 (60%), being similar to ESC after BMS implantation. In the latter (8 > DSI > 3 years), however, ESC grade varied from grade 0 (6%), grade 1 (63%), grade 2 (25%), and grade 3 (6%). Incidence of YP or ISMT was 60% in the former, suggesting progression of neoatherosclerosis at extremely late phase. In conclusion, from the viewpoint of ESC, coronary arterial repair at SES implantation appeared to catch up with BMS at around 8 years later, although incidence of YP or ISMT remained still greater in SES than BMS at extremely late phase.
Abstract 653: Development of Advanced Atherosclerotic Plaque by Injection of Inflammatory Proteins in Mini-pig Model
Authors: Lee Seulgee , Kim Jung-Sun
Published: June 2021

Objective:
Appropriate animal models of atherosclerotic plaque are crucial for the investigation of the pathophysiology of atherosclerosis, as well as for the evaluation of the efficacy and safety of vascular devices. Therefore, we aimed to develop a novel animal model that would be suitable for the study of the advanced atherosclerotic lesions in vivo.

Methods:
Atherosclerotic plaque was induced in 32 sites of 8 mini-pigs by combining a high cholesterol diet, endothelial denudation, and injection into the vessel wall with either saline or inflammatory proteins (high-mobility group protein B1 [HMGB1] and tumor necrosis factor [TNF]-α) using a Cricket™ Micro-infusion catheter. In vivo imaging with optical coherence tomography (OCT) was performed to detect the plaque characteristics at follow-up after 10 weeks. All tissues were harvested for histological evaluation.

Results:
Intima area/plaque area tended to be higher in the HMGB1, and TNF-α groups compared to the saline injected group (p=0.015). Advanced plaques were more frequently observed in the group injected with inflammatory proteins. High degree inflammation occurred in inflammatory protein injected group compared to saline injected group it immunohistochemistry stain using HMGB1 (p<0.001), RAGE (p=0.003), TNF-α antibody (p<0.001) and CD68 antibody (p=0.005). Quantitative imaging evaluation at minimal lumen diameter and percentage of diameter stenosis were also not significantly different among all group. OCT demonstrated that lipid rich plaques were more frequently detected in the inflammatory protein group (saline group: 2/11 [18%], HMGB1 group: 9/11 [82%] and TNF-α group: 5/10 [50%]).

Conclusion:
Direct injection of inflammatory proteins into the vessel wall significantly increased inflammation occurrence and induced lipid rich plaques. These data indicate that this mini-pig model of atherosclerotic lesion formation via direct injection of pro-inflammatory proteins into the vessel wall is useful for in vivo studies investigating atherosclerosis.

Key words:
mini-pig model, atherosclerosis, balloon injury, inflammatory protein
Abstract 655: Rac2 is a Key Modulator of IL-1β -dependent Atherosclerotic Plaque Calcification
Authors: Ceneri Nicolle , Zhao Lina , Young Bryan D , Healy Abigail L , Coskun Suleyman , Vasavada Hema , Yarovinsky TImur O , Ike Kenneth , Qin Lingfen , Pardi Ruggero , Meadows Judith , Tellides George , Hirschi Karen , Soufer Robert , Sadeghi Mehran , Bender Jeffrey R , Morrison Alan R
Published: June 2021

The calcium composition of atherosclerotic plaque has predictive value for increased risk of cardiovascular events. Inflammation is associated with atherosclerotic calcification, but the immune signaling that regulates calcium mineralization in plaque is minimally understood. The hematopoietic Rac family member, Rac2, modulates the activation of immune cells and has potential to influence plaque osteogenesis. Both aortic plaque from

ApoE
-/-

mice fed a high fat diet and coronary plaque from patients revealed increased Rac1:Rac2 expression ratios, driven by dynamic Rac2 expression, to be associated with calcified plaque. On high fat diet,

Rac2
-/-
ApoE
-/-

mice demonstrated comparable serum cholesterol and plaque burden relative to

ApoE
-/-

mice, but histology identified differences in plaque structure and cellularity. MicroCT and calcium-targeted imaging identified increased atherosclerotic calcification, which was associated with elevated expression of osteogenic transcription factors and was dependent on the hematopoietic compartment. Calcified plaque expressed higher IL-1β mRNA levels, and serum revealed increased IL-1β protein concentrations.

Rac2
-/-
ApoE
-/-

macrophages demonstrated increased activation of Rac1 and consequent Rac1-dependent IL-1β secretion. Downstream of Rac1, NF-κB and reactive oxygen species (ROS) signaling drove IL-1β production by increasing IL-1β mRNA expression and caspase1 activation. Cultured mouse aorta smooth muscle cells mineralized calcium in an IL-1β dose-dependent manner, and the enhanced atherosclerotic calcification
in vivo
was inhibited by IL-1 receptor antagonist, confirming a cause-and-effect relationship. In patients with stable coronary artery disease, high coronary calcium burden was associated with increased serum IL-1β, and patients with combined elevations in calcium and IL-1β had more events driven by higher mortality, reinforcing the relevance of this inflammatory calcification signaling axis to human disease. Therapeutic targeting of IL-1β expression through the balance of Rac activation has potential to impact patient care by modulating atherosclerotic calcification and consequent cardiovascular events.
Abstract 657: PDGF
versus
Fructose Dysregulation of Insulin Receptor Signaling in Vascular Smooth Muscle Cells: Pioglitazone-mediated Sensitization of Insulin Receptor Signaling is Associated with Diminished Vascular Smooth Muscle Cell Proliferation
Authors: Osman Islam , Segar Lakshman
Published: June 2021

Previous studies have shown that PDGF-induced activation of mTOR/p70S6kinase signaling is associated with dysregulation of insulin receptor substrates (IRS1 and IRS2) in vascular smooth muscle cells (VSMCs). In addition, pioglitazone (PIO, an antidiabetic drug) has been shown to inhibit mTOR/p70S6kinase signaling thereby suppressing PDGF-induced VSMC proliferation. The role of IRS1/IRS2 on proliferative signaling events has not yet been examined in VSMCs treated with fructose (an agent that induces insulin resistance) or PIO (an insulin sensitizer). In the present study, unlike PDGF treatment (48 hr) that enhanced IRS serine phosphorylation and diminished IRS2 expression, exposure of human aortic VSMCs to D-fructose (5.5 to 25 mM) for 48 hr resulted in a concentration-dependent increase in IRS1 serine phosphorylation (up to ~5-fold , n = 3), but without any changes in IRS2 protein expression. In D-fructose-treated VSMCs, Akt phosphorylation in response to acute insulin exposure (6 min) was markedly decreased (~69%, n = 3), suggesting that IRS1 serine phosphorylation alone is sufficient to dysregulate insulin receptor signaling. In addition, D-fructose treatment led to a further increase in PDGF-induced VSMC proliferation (~40%, n = 3). Furthermore, we examined the effects of PIO on PDGF-induced dysregulation of IRS in VSMCs. Notably, PIO treatment (30 μM, 48 hr) by itself led to an upregulation of IRS-2 expression (~2.5-fold) and also diminished PDGF-induced IRS1 serine phosphorylation (~63%). Under these conditions, PIO treatment resulted in significant inhibition of PDGF-induced cyclin D1 expression, Rb phosphorylation, and VSMC proliferation (~83%, n = 3). In conclusion, the present study reveals that dysregulated insulin signaling (by PDGF and fructose) is associated with enhanced VSMC proliferation, whereas insulin sensitization (by PIO) is associated with inhibition of VSMC proliferation. Together, these findings underscore the significance of intact insulin signaling toward limiting exaggerated VSMC proliferation in the vessel wall.
Abstract 659: Macrophage Beta3 Integrin Deficiency Promotes Atherosclerosis Development Through ERK-Dependent Signaling
Authors: Wolf Cristina , Damm Ellen , Isermann Berend , Semenkovich Clay , Weilbaecher Katherine , Schneider Jochen G
Published: June 2021

Atherosclerosis is a chronic inflammation of the arterial wall caused by accumulation of cholesterol. This complex process involves endothelial and smooth muscle cells as well as the recruitment and differentiation of circulating monocytes. Integrins are membrane-bound molecules that are involved in signaling between cells and the extracellular matrix, thereby influencing intracellular signaling and cytoskeletal stability. We and others have described the in vivo consequences of germline deletion of the β3 integrin in inflammation, atherosclerosis and cancer. However, the distinct pathophysiological consequences of its tissue-specific deletion in macrophage function in development of atherosclerotic lesions remain mostly elusive. Our aim was to characterize for the first time the phenotype of mice with tissue-specific deletion of β3 integrin in macrophages (LyzMCre) under high fat diet (HFD) condition on atherosclerotic prone backgrounds (ApoE-/- and LDLr-/-), and to elucidate the signaling mechanism involved using in vitro methods. Tissue specific β3 integrin deficiency in macrophages alone is sufficient to cause increased lesion formation in the aorta of mice on HFD on both backgrounds used suggesting an important atheroprotective role played by this integrin. β3 integrin-deficient bone marrow-derived macrophages (BMDM) treated with oxLDL in vitro demonstrated a strong cholesterol uptake and increased foam cell formation. We identified that β3 integrin deficiency in macrophages caused an early activation of Ras followed by ERK phosphorylation, deficient cholesterol efflux and decreased expression of cholesterol transporter (ABCA1) which is a major regulator of cellular cholesterol. Inhibition of the ERK pathway reduced foam cell formation of macrophages at least partially by restoring expression of ABCA1. Taken together, our results show that macrophage β3 integrin is an important signaling molecule for cellular activation. Its deficiency showed important functional consequences in regard to inflammation and atherosclerosis that could be possibly modulated by interfering with downstream signaling.
Abstract 660: Macrophage Specific Deficiency of the Transient Receptor Potential Canonical 3 Channel Reduces Apoptosis and Necrosis in Advanced Atherosclerotic Plaques
Authors: Solanki Sumeet A , Vazquez Guillermo
Published: June 2021

Background:
Macrophage apoptosis plays a critical role in progression of atherosclerosis. Previous studies suggest that M1 and M2 macrophage phenotypes dominate atherosclerosis. Recently, we showed that advanced lesions in the aortic root of Apoe
-/-
mice transplanted with bone marrow deficient in the calcium-permeable channel Transient Receptor Potential Canonical 3 (TRPC3) are characterized by reduced areas of necrosis and less apoptotic macrophages. However, the donor mice used in these studies had global deficiency of TRPC3, raising the question whether the observed phenotype was also contributed by TRPC3-deficient non-myeloid cells which could undermine the true impact of macrophage deletion of TRPC3. To address this important question, we generated mice with macrophage-specific loss of TRPC3 function (MacTrpc3
-/-
).

Methods & results:
13 six week-old female Ldlr
-/-
mice were irradiated and transplanted with Ldlr
-/-
(control) or MacTrpc3
-/-
Ldlr
-/-
(experimental) bone marrow and kept on high fat diet for 14 weeks. At the end of the diet period, aortic roots were sectioned and processed for atherosclerotic lesion analysis. Total lesion size (H&E), neutral lipid (Oil Red O) and macrophage content (CD68 staining) were not different between groups. However, we found a 1.7 fold decrease (P=0.01) in percent necrotic area in advanced lesions of MacTrpc3
-/-
Ldlr
-/-
mice (23.12 ± 2.07%, n=10) compared to controls (39.63 ± 5.93%, n=10). Using in situ TUNEL we found that MacTrpc3
-/-
Ldlr
-/-
lesions have less apoptotic cells compared to controls, and these overlapped with CD68
+
areas. Using iNOS and mannose receptor as markers for M1 and M2 macrophages, respectively, we found that both subsets overlapped with CD68
+
and TUNEL
+
positive areas, with no differences between groups (n=5). Previously, we showed that M1, but not M2 macrophages derived from MacTrpc3
-/-
mice, had reduced apoptosis. This suggests that reduced plaque necrosis of MacTrpc3
-/-
Ldlr
-/-
mice may be due to reduced apoptosis of M1 macrophages. In sum, these in vivo studies indicate that macrophage-specific deficiency of TRPC3 has a genuine beneficial effect on advanced atherosclerotic plaques, reducing apoptosis and necrosis, probably due to a selective effect of TRPC3 on M1 macrophages.
Abstract 661: Bilirubin Prevents Atherosclerotic Plaque Formation in LDLR
-/-
Mice by Inhibiting Monocyte Migration Through the Disruption of Endothelial Vascular Cell Adhesion Molecule 1 (VCAM-1) and Intercellular Adhesion Molecule 1 (ICAM-1) Signaling
Authors: Vogel Megan E , Idelman Gila , Zucker Stephen D
Published: June 2021

Background:
Epidemiologic studies support an inverse association between serum bilirubin levels and the risk of cardiovascular disease. Monocyte infiltration into the vascular endothelium, a critical early step in atherosclerotic plaque formation, is mediated primarily by the adhesion molecules VCAM-1 and ICAM-1. We postulate that bilirubin, a potent antioxidant, inhibits plaque formation by preventing monocyte transendothelial migration through the scavenging of VCAM-1 and ICAM-1 reactive oxygen species (ROS) signaling intermediaries.

Methods:
The effect of physiological concentrations (≤ 1.2 mg/dL) of bilirubin on the migration of THP-1 monocytes across HUVEC monolayers activated with TNFα to express VCAM-1 and ICAM-1 was assessed by Boyden chamber assay. The influence of bilirubin on monocyte adhesion, cytokine production, and VCAM-1- and ICAM-1-mediated ROS production was quantified by fluorescence assay, RT-PCR, and time-lapse confocal microscopy, respectively. To evaluate the effect on plaque formation
in vivo
, LDLR
-/-
mice were fed a Western diet and administered i.p. bilirubin (30 mg/kg), vehicle, or sham for 8 weeks. Aortic roots were stained with Oil Red O (lipids), Sirius Red (collagen), α-smooth muscle actin (SMCs), chlorotyrosine and nitrotyrosine (oxidation), CD68 (macrophages), and for VCAM-1 and ICAM-1.

Results:
THP-1 migration across activated HUVEC monolayers is dose-dependently inhibited by bilirubin. Bilirubin did not alter monocyte binding or cytokine production by activated HUVEC, but effectively abolished cellular ROS production induced by crosslinking anti-VCAM-1 and anti-ICAM-1 antibodies. Bilirubin treatment markedly inhibited plaque formation in the aortic root of LDLR
-/-
mice, and was associated with significant reductions in plaque lipid and collagen deposition, SMC infiltration, oxidation, and macrophage infiltration, without altering VCAM-1 or ICAM-1 protein expression.

Conclusion:
Bilirubin prevents atherosclerotic plaque formation in LDLR
-/-
mice by disrupting endothelial VCAM-1- and ICAM-1-mediated monocyte migration through the scavenging of ROS signaling intermediaries. These findings suggest a potential mechanism for the purported cardioprotective effects of bilirubin.
Abstract 662: Assessing the Biological Consistency in Atherosclerosis Susceptibility Genes Between Mouse and Human Genetic Studies
Authors: von Scheidt Moritz , Zhao Yuqi , Kurt Zeyneb , Schunkert Heribert , Yang Xia , Lusis Aldons J
Published: June 2021

Atherosclerosis is a complex and heritable disease with a vast number of interacting genes, networks and molecular pathways. Mouse genetics has been utilized as a powerful tool to study human atherosclerosis and has identified hundreds of candidate genes; recent human genetic studies have also identified hundreds of suggestive genetic loci for atherosclerotic coronary artery disease (CAD). However, commonalities and differences of biological mechanisms in atherosclerosis between humans and mice are poorly examined. In this study, we aim to provide a systematic biological overview comparing mice and humans on pathway level. We first comprehensively reviewed and summarized known mouse genes associated with atherosclerosis based on studies of genetically modified mouse models. This effort revealed 743 mouse atherosclerosis genes from over 9,000 Pubmed publications, retrieved using the terms “atherosclerosis” or “atherogenesis” and “gene” or “qtl” and “mouse” or “mice” and “apoe” or “ldlr”, and followed by manual review and confirmation of the effects on atherosclerosis. Second, we conducted pathway analysis of the mouse atherosclerosis genes using data from Biocarta, Kyoto Encyclopedia of Genes and Genomes (KEGG) and Reactome. We found pathways including cytokine interactions or dependency to the immune system are highly over-represented among the mouse atherosclerosis genes. Many of these pathways (e.g., metabolism of lipids and lipoproteins) converged with those reported for human GWAS loci, but also highlighted several pathways (e.g., GPCR signaling) having a hitherto unknown effect on atherosclerosis in mice. Our results suggest that although there are certain differences in individual candidate genes, mouse models remain a promising approach to identify complex molecular pathways of atherosclerosis, and insights gained from mouse genetics of atherosclerosis could be validated and transferred to CAD in humans. Future network analysis of the mouse and human genes and pathways will provide further elucidation of the shared or distinct gene-gene regulation and interactions between species. This study might provide new starting points for drug development, therapeutic treatment and prevention of atherosclerosis and CAD in humans.
Abstract 663: Modified Low Density Lipoproteins Elicited Macrophage InflammatoryResponses is Regulated by the Glycerolipid Synthesis Enzyme Lipin-1
Authors: Vozenilek Aimee E , Navratil Aaron R , Green Jonette M , Coleman David , Orr A. Wayne , Woolard Matthew D
Published: June 2021

Modified low density lipoproteins (modLDL) elicit macrophage generation into foam cells that release pro-inflammatory mediators driving atherosclerotic lesion progression causing cardiovascular disease. The molecular mechanisms that elicit foam cell inflammatory responses have not yet been fully elucidated. The lipid-laden phenotype that is characteristic of macrophage foam cells is due to lipid droplet biogenesis in response to excess cholesterol. Lipid droplet biogenesis is a process that is thought to be symptomatic of, but not drive atherosclerosis. Lipid droplet biogenesis requires glycerolipid synthesis, during which, lipin-1 converts phosphatidate into diglyceride as the penultimate step of lipid droplet generation. We had previously demonstrated lipin-1 is also required for modLDL-elicited pro-inflammatory response from macrophages. We hypothesized that modLDL elicits chronic diglyceride generation, via lipin-1 enzymatic activity, that activates signaling cascades responsible for foam cell pro-inflammatory responses. To test our hypotheses we stimulated wild type and lipin-1 depleted bone marrow-derived macrophages (BMDMs) with oxidized LDLs (oxLDLs). Stimulation of wild type BMDMs resulted in chronic activation of the signaling kinases PKCα/βII, ERK1/2 and the AP-1 transcription factor subunit cJun (up to 48 hours after stimulation). This pathway was not observed to be active in BMDMs depleted of lipin-1 either genetically or with siRNA. The pharmacological inhibition of lipin-1, PKCα/βII, ERK1/2 strongly suggest lipin-1- PKCα/βII-ERK1/2-cJun represents a signaling axis. Finally, each of these proteins were required for oxLDL-elicited pro-inflammatory responses by macrophages. These results suggest that augmented glycerolipid synthesis in macrophages due to modLDL stimulation is not just symptomatic of atherosclerosis but promote inflammatory responses that drive lesion progression
Abstract 664: Effects of Eicosapentaenoic Acid Plus Docosapentaenoic Acid and Eicosapentaenoic Acid Alone on Fasting and Postprandial Monocyte Phenotypes
Authors: Wu Huaizhu , Perrard Xiao-Yuan D , Bobotas George , Maki Kevin C , Ballantyne Christie M
Published: June 2021

Background and objective:
Fasting and postprandial (PP) hypertriglyceridemia is associated with increased lipid accumulation in circulating monocytes and monocyte phenotypic changes, e.g., increased expression of CD11c (a β2 integrin), which may increase risk for atherosclerosis. Omega-3 fatty acids lower circulating triglycerides (TG). This study examined whether treatment with omega-3 fatty acids improves fasting and PP monocyte phenotypes associated with hypertriglyceridemia.

Methods:
This was an open-label, randomized, crossover study of men and women with elevated TG (200-400 mg/dL) to evaluate the effects of MAT9001 (eicosapentaenoic acid [EPA] plus docosapentaenoic acid [DPA], Matinas BioPharma Inc., USA), compared to Vascepa (EPA-ethyl esters [EE] only, Amarin Pharma Inc., USA), administered at 4 g/d dosages for 15 d (≥28-d washout between treatments). Fasting and PP (4 and 6 h) TG and monocyte phenotypes (analyzed by flow cytometry) were examined prior to and on d 15 of each treatment.

Results:
MAT9001 and Vascepa lowered fasting TG from baseline (-32% and -19% respectively; both p≤0.04; n=26-29). Only MAT9001 significantly reduced PP TG area under the curve (AUC) from pre- to post-treatment (p<0.01). The reduction in TG AUC was greater for MAT9001 (-23%) vs. Vascepa (+4%) (p=0.01). Treatment with MAT9001 or Vascepa reduced fasting and PP CD11c mean fluorescent intensity (MFI) levels on classical (cM) and intermediate (iM) monocytes by 6.3-23.2% (no significant differences between treatments; Table). Only MAT9001 significantly reduced PP CD11c MFI on nonclassical monocytes (ncM) and lowered fasting nile red levels, indicating less lipid accumulation, in cM and iM (Table).

Summary:
Decreases in TG produced by MAT9001 or Vascepa in men and women with hypertriglyceridemia were associated with reduced fasting and PP CD11c on cM and iM. MAT9001, but not Vascepa, significantly reduced PP ncM CD11c and fasting cM and iM nile red staining for lipids.
Abstract 665: High-monounsaturated Fat Diet Lowers Foamy Monocyte Formation in ApoE-deficient Mice
Authors: Perrard Xiao-Yuan D , Wu Huaizhu
Published: June 2021

Intake of western diet (WD) high in saturated fat and cholesterol causes formation of foamy monocytes (FMs; monocytes containing intracellular lipid droplets) in the circulation, which contribute to atherogenesis, in apoE-/- mice. It remains to be determined which diet component, fat or cholesterol, is more important in inducing FM formation. Mediterranean-type diet (MedD) high in monounsaturated fat (MUF) protects against atherosclerotic events. It is unknown whether MedD affects FM formation. To address these questions, 4 groups of apoE-/- mice were randomly assigned to the following diets: 1) WD (21% w/w milkfat containing 13.3% saturated fat and 5.9% MUF; 0.2% w/w cholesterol); 2) high-fat low-cholesterol diet (HFD, 21% milkfat; 0.05% cholesterol); 3) high-cholesterol low-fat diet (HCD, 5% milkfat; 0.2% cholesterol); 4) MedD with high cholesterol (21% fat containing 2.6% saturated fat and 13.4% MUF from 15% extra-virgin olive oil, 3% walnut fat, 1.5% almond fat and 1.5% hazelnut fat; 0.2% cholesterol). FMs and phenotypes were examined by flow cytometry. Calorie intake and weight changes were not different among the 4 groups of mice. At 1 week on diets, mice on WD or HCD, but not mice on HFD, had abundant FMs in the circulation (Table). Compared to mice on WD or HCD, mice on MedD had lower proportions of FMs and lower side scatter (SSC) values, indicating less lipid, in FMs. Mean fluorescence intensity of CD36, a scavenger receptor, tended to be lower on FMs in mice on MedD than in mice on WD. At 8 weeks, mice on HFD also had abundant FMs, which were, however, still lower than those in mice on WD. Compared to those in mice on WD, the percentage and SSC values of FMs remained lower in mice on MedD for 8 weeks (Table). In summary, high cholesterol appears to be the major dietary component that induces early FM formation in apoE-/- mice while persistent intake of high saturated fat also induces FM formation. Fewer FMs in mice on MedD than on WD or HCD suggests that MUF in MedD may inhibit FM formation.
Abstract 96: The Role of Flavin Monooxygenase 3 (FMO3) in Dietary Choline- and Cholesterol-Driven Atherosclerosis
Authors: Schugar Rebecca C , Gromovsky Anthony D , Brown Amanda L , Burrows Amy C , Ferguson Daniel L , Graham Mark J , Crooke Rosanne M , Lee Richard G , Wang Zeneng , Hazen Stanley L , Brown J. Mark
Published: June 2021

The gut microbiome represents an important endocrine organ generating nutrient metabolites that promote the pathogenesis of cardiovascular disease (CVD), as exemplified by the gut microbe-derived metabolite trimethylamine-N-oxide (TMAO). The meta-organismal TMAO pathway is initiated by ingestion of nutrients present in high fat foods (phosphatidylcholine, choline, and
L
-carnitine), which are metabolized by gut microbes to generate trimethylamine (TMA). The gut microbial metabolite TMA is further metabolized by the host enzyme flavin monooxygenase 3 (FMO3) to produce TMAO, which signals to drive proatherogenic programs in the host. Our previous work has demonstrated that the TMAO-producing enzyme FMO3 is a central regulator of whole body cholesterol balance and reverse cholesterol transport (RCT). However, the ability of FMO3 to regulate cholesterol balance and RCT is largely dependent on the amount of dietary cholesterol available. Given that the meta-organismal TMAO pathway is highly dependent on dietary cues, we hypothesized that the host enzyme FMO3 may be a central integrator of both choline- and cholesterol-driven atherosclerosis. To determine the role of FMO3 in diet-driven atherosclerosis progression, female hyperlipidemic LDLr
-/-
mice received either control or FMO3 antisense oligonucleotides (ASO) while being fed a basal control diet or proatherogenic diets containing 1) 0.2% cholesterol, 2) 1.2% choline, or 3) both 0.2% cholesterol and 1.2% choline for 16 weeks. FMO3 knockdown markedly reduced plasma cholesterol under all dietary conditions, yet only reduced plasma triglycerides in the choline-supplemented mice. Unexpectedly, FMO3 knockdown also elevated circulating monocytes and altered T cell subsets in a diet-specific manner, indicating a role for FMO3 in immune cell maturation only when certain nutrients are available in the diet. The relationship to these diet- and FMO3-dependent CVD risk factors to total atherosclerosis lesion area and lesion-associated immune cell burden will also be presented. Collectively, our studies suggest that host enzyme FMO3 plays a central role in integrating dietary cues (choline & cholesterol) to lipid metabolic and immune cell reprogramming that synergize to promote atherosclerosis.
Obstructive Sleep Apnea and Cardiovascular Disease: A Scientific Statement From the American Heart Association
Authors: Yeghiazarians Yerem , Jneid Hani , Tietjens Jeremy R. , Redline Susan , Brown Devin L. , El-Sherif Nabil , Mehra Reena , Bozkurt Biykem , Ndumele Chiadi Ericson , Somers Virend K. ,   on behalf of the American Heart Association Council on Clinical Cardiology; Council on Peripheral Vascular Disease; Council on Arteriosclerosis, Thrombosis and Vascular Biology; Council on Cardiopulmonary, Critical Care, Perioperative and Resuscitation; Stroke Council; and Council on Cardiovascular Surgery and Anesthesia
Published: June 2021

Obstructive sleep apnea (OSA) is characterized by recurrent complete and partial upper airway obstructive events, resulting in intermittent hypoxemia, autonomic fluctuation, and sleep fragmentation. Approximately 34% and 17% of middle-aged men and women, respectively, meet the diagnostic criteria for OSA. Sleep disturbances are common and underdiagnosed among middle-aged and older adults, and the prevalence varies by race/ethnicity, sex, and obesity status. OSA prevalence is as high as 40% to 80% in patients with hypertension, heart failure, coronary artery disease, pulmonary hypertension, atrial fibrillation, and stroke. Despite its high prevalence in patients with heart disease and the vulnerability of cardiac patients to OSA-related stressors and adverse cardiovascular outcomes, OSA is often underrecognized and undertreated in cardiovascular practice. We recommend screening for OSA in patients with resistant/poorly controlled hypertension, pulmonary hypertension, and recurrent atrial fibrillation after either cardioversion or ablation. In patients with New York Heart Association class II to IV heart failure and suspicion of sleep-disordered breathing or excessive daytime sleepiness, a formal sleep assessment is reasonable. In patients with tachy-brady syndrome or ventricular tachycardia or survivors of sudden cardiac death in whom sleep apnea is suspected after a comprehensive sleep assessment, evaluation for sleep apnea should be considered. After stroke, clinical equipoise exists with respect to screening and treatment. Patients with nocturnally occurring angina, myocardial infarction, arrhythmias, or appropriate shocks from implanted cardioverter-defibrillators may be especially likely to have comorbid sleep apnea. All patients with OSA should be considered for treatment, including behavioral modifications and weight loss as indicated. Continuous positive airway pressure should be offered to patients with severe OSA, whereas oral appliances can be considered for those with mild to moderate OSA or for continuous positive airway pressure–intolerant patients. Follow-up sleep testing should be performed to assess the effectiveness of treatment.
Abstract 003: Personalizing the Intensity of Blood Pressure Control: Modeling the Heterogeneity of Risks and Benefits From the SPRINT Trial
Authors: Patel Krishna K , Arnold Suzanne V , Chan Paul S , Tang Yuanyuan , Pokharel Yashashwi , Jones Philip G , Spertus John A
Published: June 2021

Introduction:
In SPRINT (Systolic blood PRessure INtervention Trial), non-diabetic patients with hypertension at high cardiovascular risk treated with intensive blood pressure (BP) control (<120mmHg) had fewer major adverse cardiovascular events (MACE) and all-cause deaths but higher rates of serious adverse events (SAE) compared with patients treated with standard BP control (<140mmHg). However, the degree of benefit or harm for an individual patient could vary due to heterogeneity in treatment effect.

Methods:
Using patient-level data from SPRINT, we developed predictive models for benefit (freedom from death or MACE) and harm (increased SAE) to allow for individualized BP treatment goals based on projected risk-benefit for each patient. Interactions between candidate variable and treatment were evaluated in the models to identify differential treatment effects. We performed 10 fold cross-validation for both the models.

Results:
Among 9361 patients, 8606 (92%) patients had no MACE or death event (benefit) and 3529 (38%) patients had a SAE (harm) over a median follow-up of 3.3 years. The benefit model showed good discrimination (c-index= 0.72; cross-validated c-index= 0.72) with treatment interactions of age, sex, and baseline systolic BP (Figure A), with more benefit of intensive BP treatment in patients who are older, male, and have lower baseline SBP. The SAE risk model showed moderate discrimination (c-index=0.66; cross-validated c-index= 0.65) with a treatment interaction of baseline renal function (Figure B), indicating less harm of intensive treatment in patients with a higher baseline creatinine. The mean predicted absolute benefit of intensive BP treatment was of 2.2% ± 2.5% compared with standard treatment, but ranged from 10.7% lower benefit to 17% greater benefit in individual patients. Similarly, mean predicted absolute harm with intensive treatment was 1.0% ± 1.9%, but ranged from 15.9% lesser harm to 4.9% more harm.

Conclusion:
Among non-diabetic patients with hypertension at high cardiovascular risk, we developed prediction models using basic clinical data that can identify patients with higher likelihood of benefit vs. harm with BP treatment strategies. These models could be used to tailor the treatment approach based on the projected risk and benefit for each unique patient.
Improving the Effectiveness of Psychological Interventions for Depression and Anxiety in Cardiac Rehabilitation: PATHWAY—A Single-Blind, Parallel, Randomized, Controlled Trial of Group Metacognitive Therapy
Authors: Wells Adrian , Reeves David , Capobianco Lora , Heal Calvin , Davies Linda , Heagerty Anthony , Doherty Patrick , Fisher Peter
Published: June 2021

Background:

Depression and anxiety in cardiovascular disease are significant, contributing to poor prognosis. Unfortunately, current psychological treatments offer mixed, usually small improvements in these symptoms. The present trial tested for the first time the effects of group metacognitive therapy (MCT; 6 sessions) on anxiety and depressive symptoms when delivered alongside cardiac rehabilitation (CR).

Methods:

A total of 332 CR patients recruited from 5 National Health Service Trusts across the North-West of England were randomly allocated to MCT+CR (n=163, 49.1%) or usual CR alone (n=169, 50.9%). Randomization was 1:1 via minimization balancing arms on sex and Hospital Anxiety and Depression Scale scores within hospital site. The primary outcome was Hospital Anxiety and Depression Scale total after treatment (4-month follow-up). Secondary outcomes were individual Hospital Anxiety and Depression Scales, traumatic stress symptoms, and psychological mechanisms including metacognitive beliefs and repetitive negative thinking. Analysis was intention to treat.

Results:

The adjusted group difference on the primary outcome, Hospital Anxiety and Depression Scale total score at 4 months, significantly favored the MCT+CR arm (–3.24 [95% CI, –4.67 to –1.81],
P
<0.001; standardized effect size, 0.52 [95% CI, 0.291 to 0.750]). The significant difference was maintained at 12 months (–2.19 [95% CI, –3.72 to –0.66],
P
=0.005; standardized effect size, 0.33 [95% CI, 0.101 to 0.568]). The intervention improved outcomes significantly for both depression and anxiety symptoms when assessed separately compared with usual care. Sensitivity analysis using multiple imputation of missing values supported these findings. Most secondary outcomes favored MCT+CR, with medium to high effect sizes for psychological mechanisms of metacognitive beliefs and repetitive negative thinking. No adverse treatment-related events were reported.

Conclusions:

Group MCT+CR significantly improved depression and anxiety compared with usual care and led to greater reductions in unhelpful metacognitions and repetitive negative thinking. Most gains remained significant at 12 months. Study strengths include a large sample, a theory-based intervention, use of longer-term follow-up, broad inclusion criteria, and involvement of a trials unit. Limitations include no control for additional contact as part of MCT to estimate nonspecific effects, and the trial was not intended to assess cardiac outcomes. Nonetheless, results demonstrated that addition of the MCT intervention had broad and significant beneficial effects on mental health symptoms.

Registration:

URL:
https://www.clinicaltrials.gov
; Unique identifier: ISRCTN74643496.
Abstract 078: Postnatal Treatment with Metyrapone Attenuates the Effects of Early Life Stress (ELS) on Diet-induced Obesity in Female Rats
Authors: Murphy Margaret O , Cohn Dianne M , Loria Analia S
Published: June 2021

Clinical studies have shown a positive correlation between ELS and the development of cardiometabolic disease, particularly affecting women. We previously reported that male rats exposed to Maternal Separation (MatSep), a model of ELS in rodents, do not develop exaggerated diet-induced obesity. Thus, this study tested the hypothesis that MatSep exacerbates the response to an obesogenic diet in female rats. Also, we tested whether the postnatal treatment with metyrapone (MTP), a corticosterone synthase inhibitor, would attenuate this phenotype. MatSep was performed in WKY offspring during 3 hours/day from postnatal days 2-14. Non-disturbed littermates were used as controls. Female rat offspring were untreated or treated with MTP (50 mg/kg, i.p.), 30 minutes prior the daily separation. Upon weaning, rats were placed on regular chow (ND, 18% kcal fat) or HFD (60% kcal fat) for 12 weeks. Despite no differences in food intake (metabolism cages) and blood pressure (DSI radiotelemetry) MatSep exaggerated body weight gain and fat pad weights (p<0.05) in response to HFD. Also, MatSep increased plasma corticosterone (189±48 vs. 79±18 pg/ml, p<0.05) and leptin (2.1±0.4 vs. 1.5±0.4 ng/ml, p<0.05) levels compared to control while insulin and adiponectin levels were similar between groups. Oral glucose tolerance test was impaired in MatSep rats showing a greater AUC compared to control rats (p<0.05). Importantly, MTP-treated female MatSep rats showed significantly attenuated diet-induced obesity, glucose intolerance, plasma corticosterone and leptin levels. Histological analysis revealed that MTP treatment ameliorated the adipocyte size in visceral fat from MatSep rats as well (p<0.05). Gene expression in liver indicated that glucose 6 phospatase, but not other gluconeogenic enzymes, were increased in obese MatSep rats, whereas the MTP treatment abrogated this effect. The visceral fat gene expression showed lower levels of insulin receptor in MatSep rats, but no differences in other genes related to the glucose disposal. Overall, these data reveal that female MatSep rats display a greater susceptibility for the synergistic effect between obesogenic diet and ELS compared to male rats, and this effect may be linked to early life exposure to stress hormones.
Metabolomics Analysis Reveals Deranged Energy Metabolism and Amino Acid Metabolic Reprogramming in Dogs With Myxomatous Mitral Valve Disease
Authors: Li Qinghong , Larouche‐Lebel Éva , Loughran Kerry A. , Huh Terry P. , Suchodolski Jan S. , Oyama Mark A.
Published: June 2021

Background

Myxomatous mitral valve disease (MMVD), a naturally occurring heart disease, affects 10% to 15% of the canine population. Canine MMVD shares many similarities with human MMVD. Untargeted metabolomics was performed to identify changes in metabolic pathways and biomarkers with potential clinical utilities.

Methods and Results

Serum samples from 27 healthy, 22 stage B1, 18 stage B2 preclinical MMVD dogs, and 17 MMVD dogs with a history of congestive heart failure (CHF) were analyzed. Linear regression analysis identified 173 known metabolites whose concentrations were different among the 4 groups (adjusted
P
<0.05), of which 40% belonged to amino acid super pathways, while 30% were lipids. More than 50% of significant metabolites were correlated with left atrial diameter but not left ventricular dimension. Acylcarnitines, tricarboxylic acid cycle intermediates, and creatine accumulated in proportion to MMVD severity. α‐Ketobutyrate and ketone bodies were increased as MMVD advanced. Nicotinamide, a key substrate of the main nicotinamide adenine dinucleotide (NAD
+
) salvage pathway, was decreased, while quinolinate of the de novo NAD
+
biosynthesis was increased in CHF dogs versus healthy dogs. 3‐Methylhistidine, marker for myofibrillar protein degradation, was higher in CHF dogs than non‐CHF dogs. Trimethylamine N‐oxide (TMAO) and TMAO–producing precursors, including carnitine, phosphatidylcholine, betaine, and trimethyllysine, were increased in CHF dogs versus non‐CHF dogs. Elevated levels of uremic toxins, including guanidino compounds, TMAO, and urea, were observed in CHF dogs. Pathway analysis highlighted the importance of bioenergetics and amino acid metabolism in canine MMVD.

Conclusions

Our study revealed altered energy metabolism, amino acid metabolic programming, and reduced renal function in the development of MMVD and CHF. Complex interplays along the heart‐kidney‐gut axis were implicated.