bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 439: Atherosclerosis Development is Reduced in Mice With Blunted Biliary Cholesterol Secretion
Authors: Cai Lei , Layne Joseph D , Paxton Sierra M , Burkett Courtney R , Lee Richard , Temel Ryan E
Published: June 2021

Excessive accumulation of cholesterol in the arteries drives atherosclerosis development. It is believed that biliary cholesterol secretion is crucial for eliminating excess cholesterol from the body via reverse cholesterol transport. In the current study, we wanted to determine the impact of reduced biliary cholesterol secretion on atherosclerosis development in mice. Decreased biliary cholesterol secretion was achieved by hepatic over-expression of human NPC1L1 (L1Tg mice) combined with knockdown of hepatic ABCG5/G8 function using an ABCG8 antisense oligonucleotide (ASO). LDLR-/- and LDLR-/-/L1Tg mice received either control or ABCG8 ASO and were fed a high fat (42% Kcal)/low cholesterol diet (0.015% wt/wt) for 20 weeks. As expected, L1Tg mice and mice with hepatic ABCG8 knockdown had an >70% reduction in biliary cholesterol. The dramatic decrease in biliary cholesterol did not increase plasma cholesterol, and in fact mice with hepatic ABCG8 knockdown had reduced VLDL cholesterol. Even more surprising, aortic atherosclerosis was significantly decreased in mice with compromised biliary cholesterol secretion. LDLR-/-/L1Tg treated with ABCG8 ASO had a >90% reduction in biliary cholesterol yet had ~70% less atherosclerosis compared to LDLR-/- controls. Moreover, reducing biliary cholesterol had no impact on macrophage reverse cholesterol transport, fecal excretion of neutral sterol, and hepatic expression of genes involved in cholesterol synthesis (HMG CoA reductase/synthase) and HDL metabolism (ABCA1 and SR-BI). These results indicate that atherosclerosis development can be decreased by shunting cholesterol away from biliary secretion and potentially towards trans-intestinal cholesterol excretion or bile acid synthesis.
Abstract 444: Semaphorin3A Reduces Atherosclerotic Plaque Formation in ApoE Knock Out Mice Through Regulation of M2 Type Macrophage Migration
Authors: Ebrahimian Talin , Heidari Maryam , Simon David , Vali Hojatollah , Mandato Craig A , Lehoux Stephanie
Published: June 2021

Objective:
Recent data point to an important immunomodulatory role for neural guidance molecules, including semaphorin 3a (Sema-3A), in inflammatory diseases. Sema-3A is a secreted member of the Sema family and is produced by several immune cells including macrophages. We found that Sema-3A receptors are expressed in macrophages and in atherosclerotic plaques.

Approach and results:
To investigate the role of Sema-3A in atherosclerosis, ApoE-/- mice were administered with a Sema-3A overexpressing or a control plasmid and were fed a high fat diet for 9 weeks. Sema-3A receptors were expressed in macrophages and atherosclerotic plaques. Our results show that Sema-3A overexpressing mice had significantly smaller atherosclerotic plaques than control mice in the aortic sinus (0.3±0.02 vs. 0.4±0.03 mm
2
), the brachiocephalic artery (0.04±0.01 vs. 0.1±0.01 mm
2
) and the aorta (9.5±1.4 vs 15.3±2.9%), assessed by oil red O staining. No differences were observed in plaque stability, measured by collagen and smooth muscle cell alpha-actin staining. However, there was significantly less (2-fold) macrophage content in the plaques of Sema-3A compared to control mice, associated with decreased circulating monocytes determined by flow cytometry as cd11b positive and Gr-1 negative cells (4.97±0.74 vs. 7.2±0.62%). To better define the involved mechanisms, we investigated macrophage function
In vitro
and found that recombinant Sema-3A increased by 4 fold migration of M2 but not M1 macrophages. In addition, active beta-1 integrin expression was significantly enhanced (2-fold) by Sema-3A in human M2 macrophages. Importantly, Sema-3A induced a significant increase (by 50%) of focal adhesion kinase phosphorylation.

Conclusions:
Our data show that Sema-3A prevents atherosclerotic plaque formation in ApoE
-/-
mice. This may be due in part to enhanced motility and function of M2 macrophages through regulation of beta-1 integrin.
Abstract 45: Sortilin Regulates Arterial Calcification in Atherosclerotic Mice and Associates With Cardiovascular Risk in Humans
Authors: Goettsch Claudia , Hutcheson Joshua , Iwata Hiroshi , Hagita Sumihiko , Libby Peter , Aikawa Masanori , Szulc Pawel , Aikawa Elena
Published: June 2021

Background:
Arterial calcification associates with cardiovascular morbidity and mortality. Human genome-wide association studies linked the SORT1 gene encoding sortilin and coronary artery calcification. We previously demonstrated
in vitro
that calcification requires sortilin phosphorylation by Fam20C. We hypothesized that sortilin mediates arterial calcification
in vivo
and that serum sortilin levels can predict cardiovascular events.

Results:
We assessed the effects of genetic deletion of sortilin (
Sort1
) in LDL receptor-deficient mice (
Ldlr
-/-
) consuming a high-fat, high-cholesterol diet.
Sort1
-/-
Ldlr
-/-
mice exhibited 80% lower aortic calcification as compared to control
Ldlr
-/-
mice in intact arteries (fluorescence reflectance imaging of a NIRF calcium tracer, p<0.01).
Sort1
-/-
Ldlr
-/-
mice showed 88% lower extracted aortic calcium (p<0.05). Histological analysis revealed less tissue non-specific alkaline phosphatase activity and arterial microcalcification. Transfer of
Sort1
-deficient bone marrow cells to irradiated atherosclerotic mice does not alter aortic calcification, indicating that immune cell-derived sortilin may not contribute to sortilin-mediated arterial calcification. In contrast to arterial calcium,
Sort1
-deficiency did not alter bone mineralization assessed by μCT and histomorphometry. In a community-based cohort of men aged ≥50 (n=830), abdominal aortic calcification (AAC) was assessed by dual energy X-ray absorptiometry using Kauppila score. Serum sortilin levels were measured by ELISA. After adjustment for confounders (including cholesterol concentration), sortilin levels correlated positively with severe AAC. The highest sortilin quartile associated with higher odds of severe AAC even in men without ischemic heart disease (OR=2.0, p<0.05). Among 745 men followed up prospectively for 7.8 years, major adverse cerebrovascular events occurred in 76 men. Elevated serum sortilin associated with higher event risk (adjusted for confounders, HR=2.8, p<0.001) even in men without diabetes (HR=3.5, p<0.001).

Conclusions:
Sortilin is a novel therapeutic target to modulate the formation of arterial calcification and a biomarker of both aortic calcification and cardiovascular risk.
Abstract 465: Lipid-lowering Agent Gemcabene Down-regulates Acute Phase C-reactiveProtein via /EBP-δ-mediated Transcriptional Mechanism and Attenuates Inflammation and Osteoarthritis in Animal Models
Authors: Srivastava Rai Ajit K , Cornicelli Joseph A , Markham Bruce E , Bisgaier Charles L
Published: June 2021

Background:
Inflammation plays a key role in setting the stage as well as causing the progression of atherosclerosis. High sensitivity c-reactive protein (hsCRP), an acute phase reactant released during inflammatory processes, has been recognized as a predictor of cardiovascular risk.

Experiments & Results:
Since gemcabene reduced hsCRP in humans, we investigated the mechanism of hsCRP reduction and efficacy of anti-inflammatory activity in animal models of arthritis and pain. In human hepatoma cell line, PLC/PRF/5, gemcabene showed dose-dependent inhibition of IL-6+ IL-1β-induced CRP production by -70% inhibition at 2 mM. In TNF-α-stimulated primary human coronary artery endothelial cells, both CRP and IL-6 productions were inhibited by gemcabene in a dose-dependent manner (-70% at 2mM). Transfection studies with human CRP regulatory sequences in luciferase/ β-gal system showed a 25-fold increase in IL-6- as well as IL-6+ IL-1β -stimulated CRP transcription, which was reduced by gemcabene (-50% at 2 mM), suggesting transcriptional down-regulation of CRP. Site-directed mutation of C/EBP, NF-κB, and STAT sites of the human CRP promoter suggested that the overlapping downstream C/EBP and NF-κB binding sites are important for gemcabene-mediated down-regulation of CRP promoter. STAT3 response element, while needed for IL-6-induced expression of CRP, is not required for gemcabene-mediated inhibition. Identification of the protein, in a gel-shift assay, that interacts with C/EBP binding sites revealed it to be C/EBPδ. Anti-inflammatory efficacy of gemcabene was evaluated in a rat model of monosodium iodoacetate (MIA)-induced osteoarthritis (OA) and carrageenan-induced thermal hyperalgesia (CITH). Gemcabene improved joint comfort (-50% at 30 mg/kg/d for 2 wk) in MIA and attenuated paw withdrawal latency (60% at 30 mg/kg/d and 97% at 100 mg/kg/d, compared to untreated control) in the CITH model. These findings were further confirmed by an IL-6/IL-6sR knee injection model showing 63 and 71% reduction in hind paw weight distribution at 10 and 30 mg/kg/d doses, respectively.

Conclusions:
Gemcabene decreases CRP by C/EBPδ and NF-κB mediated transcriptional mechanism, and attenuates inflammation-induced OA and hyperalgesia.
Abstract 470: Outpatient Community Based Educational Program Reduces Lipids in Two Weeks
Authors: Ramirez Francisco E , Siebold Jill , Antuna Kathelyn , Sanchez Albert , Nedley Neil
Published: June 2021

Background:
Dyslipidemias are a common problem among Caucasians. Community based lifestyle education programs could be an effective tool to manage lipids.

Hypothesis:
A community based program could have a positive influence in diet, habits and blood lipids.

Study Methods:
A group of 23 Caucasian retirees from Canada and America who spend their winters in San Felipe, Baja California, Mexico, participated in a two week program. No food was provided, only educational principles. The town where the program took place is known for having very poor access to fresh produce. This study included an educational component of classes twice a week on diet and lifestyle instruction, plant based cooking classes, menu planning and recipe book distribution. Each participant was evaluated via a food frequency questionnaire. Blood tests were taken at baseline and at the end of the study to measure the fasting serum lipid levels. Laboratory results were reported in mg/dL.

Results:
From 23 participants (14 females), 19 completed the two-week plant based diet trial. All participants decreased their total cholesterol. Laboratory results at the beginning of the study reported an initial mean, median, mode total cholesterol of 224, 219, 173 (SD 49, SEM 10.2), the initial mean, median, mode LDL cholesterol was 147, 136, 106 (SD 52, SEM 10.9), the initial mean, median, mode HDL cholesterol was 44, 42, 35 (SD 11, SEM 2.2), and the initial mean triglyceride level was 140 (SD 53, SEM 11.3). At the completion of the program the mean, median, mode total cholesterol was 177, 176, 145 (SD 31, SEM 7.1), the end mean, median, mode LDL cholesterol was 122, 124, N/A, (SD 24, SEM 5.5), the end mean, median, mode HDL cholesterol was 36, 35, 35 (SD 10, SEM 2.3), and the end mean triglyceride level was 128 (SD 45, SEM 10.4). The average total cholesterol decreased 21%, LDL decreased 25%, HDL decreased 17% and triglycerides decreased 8%.

Conclusion:
Even in areas where there is poor produce, a plant based diet is a good way of helping participants with dyslipidemia in a short amount of time. A long term follow up would be advised.
Abstract 48: Administration of Gut Bacteria Expressing N-acyl Phosphatidylethanolamine Reduces Steatohepatitis in LDLR-/- Mice Fed a Western Diet
Authors: Zhang Linda S , Chen Zhongyi , Zhang Youming , Ding Lei , Yancey Patricia G , Kennedy Arion , Linton MacRae F , Hasty Alyssa , Davies Sean S
Published: June 2021

Background:
The rise in obesity in the United States has led to a concomitant rise in prevalence of non-alcoholic fatty liver disease (NAFLD). The four stages of NAFLD include accumulation of triglyceride (hepatosteatosis), development of chronic inflammation (non-alcoholic steatohepatitis, NASH), fibrosis, and finally cirrhosis. Unlike wildtype C57BL6 mice, low density lipoprotein receptor (LDLR)
-/-
mouse fed a diet enriched in fat and cholesterol (Western Diet) progress to NASH and fibrotic stages of NAFLD. We showed that incorporating engineered bacteria expressing N-acyl phosphatidylethanolamine (NAPE) into the gut microbiota can inhibit development of obesity. NAPE is a precursor of N-acylethanolamines, which are bioactive lipids with anti-inflammatory functions. Here, we test the hypothesis that administering these NAPE-expressing bacteria inhibits development of NASH and fibrosis.

Methods:
NAPE-expressing E. coli Nissle 1917 (pNAPE-EcN, n=10), control Nissle 1917 (pEcN, n=10), or vehicle (veh, n=10) were given via drinking water to LDLR
-/-
mice fed a Western diet for 12 weeks. LDLR
-/-
mice fed a low fat diet (LFD) (n=10) were included for comparison.

Results:
pNAPE-EcN reduced adiposity by 26% compared with pEcN and veh (P<0.05). pNAPE-EcN also dramatically reduced hepatic triglyceride levels by 45% (p<0.05) and lipid droplet size, as well as the hepatic expressions of tissue necrosis factor α (TNFα, p<0.05), chemokine receptor 2 (CCR2, p<0.01), and tissue inhibitor of matrix metalloproteinase (TIMP1, p<0.05), consistent with reduced NASH and fibrosis. Sirius Red staining of liver sections further demonstrated reduced fibrosis. Because fatty liver is associated with atherosclerosis, we checked to see if pNAPE-EcN was able to reduce the development of atherosclerotic lesions. While serum cholesterol was reduced by 23% with pNAPE-EcN treatment (p<0.05), atherosclerotic lesion size in proximal or en face aortas only tended to be reduced (20%, 18.6%) but was not statistically significant.

Conclusions:
Our results demonstrate that incorporating therapeutically modified bacteria into the gut microbiota has potential to inhibit the development of NAFLD.
Abstract 507: Change in Pulse Wave Velocity and Short Term Development of Cardiovascular Events in the Hemodialysis Population
Authors: Daaboul Yazan , Korjian Serge , El-Ghoul Balsam , Samad Salam , Salameh Pascale , Dahdah Georges , Hariri Essa , Mansour Anthony , Spielman Kathryn , Blacher Jacques , Safar Michel , Aoun Bahous Sola
Published: June 2021

Objectives:
It has previously been demonstrated that single elevated measurements of carotid-femoral pulse wave velocity (CFPWV) among end-stage renal disease (ESRD) patients are associated with an increased risk of cardiovascular (CV) events. However, the association between single measurements of CFPWV and CV events was mostly driven by the high incidence of late events beyond 12 months of follow-up. The present prospective study aims to compare single measurements of CFPWV vs. the 2-year change in CFPWV (delta CFPWV) and their association with short-term development of CV events through 12 months in hemodialysis patients.

Methods:
Patients on hemodialysis were enrolled for a first measurement of CFPWV (CFPWV
t=0
) followed by a second measurement (CFPWV
t=1
) 2 years ± 1 month later. Patients were then followed up for an additional 12 months for the development of CV events (CV death, acute coronary syndrome, acute ischemic peripheral event necessitating intervention, or stroke).

Results:
Both CFPWV
t=0
and CFPWV
t=1
were measured for a total of 66 hemodialysis patients. During the 12-month follow-up period, 13 patients developed a total of 15 events, almost half (7/15) of which were fatal, 4/15 attributed to acute coronary syndrome (ACS), and 4/15 attributed to acute ischemic peripheral artery disease (PAD) necessitating intervention. No stroke events were recorded over the 12-month follow-up duration. Delta CFPWV (CFPWV
t=1
- CFPWV
t=0
) was significantly associated with the development of CV events through 12 months with and without adjustment for CV risk factors, including blood pressure parameters (unadjusted: HR=1.22; p=0.001 vs. adjusted: HR=1.14; p=0.002). When delta CFPWV was substituted for either CFPWV
t=0
or CFPWV
t=1
in both the unadjusted and adjusted Cox regression models, neither measurement was associated with the development of new CV events.

Conclusions:
The change in CFPWV, but not single measurements of CFPWV, was significantly associated with the short-term development of CV events through 12 months.
Abstract 512: The Long Non-coding Rna MIAT Regulates Smooth Muscle Cell Proliferation and Macrophage Activity in Advanced Atherosclerotic Lesions
Authors: Li Yuhuang , Jin Hong , Perisic Ljubica , Chernogubova Ekaterina , Bäcklund Alexandra , Gustafsson Peter , Werngren Olivera , Busch Albert , Eken Suzanne , Sun Changyan , Hansson Göran K , Paulsson-Berne Gabrielle , Hedin Ulf , Bergmark Claes , Maegdefessel Lars
Published: June 2021

Background:
Long noncoding RNAs (lncRNAs) have emerged as critical epigenetic regulators in various biological processes and diseases. Here we sought to identify and functionally characterize the lncRNA MIAT as a novel regulator in atherosclerotic plaque stability.

Methods and results:
We profiled RNA transcript expression in patients with advanced atherosclerotic lesions from the Biobank of Karolinska Endarterectomies (BiKE). By microarray analysis, lncRNA MIAT was identified as one of the most highly up-regulated non-coding RNAs in carotid plaques compared to iliac artery controls, which was confirmed by qRT-PCR and in situ hybridization. Additional in silico analysis indicated a substantial positive correlation of MIAT with markers of inflammation, apoptosis and matrix degradation in carotid plaques. Experimental knock-down of MIAT, utilizing site-specific antisense oligonucleotides (LNA-GapmeRs) not only markedly decreased proliferation and migration rates of cultured human carotid artery smooth muscle cells (hCASMCs), but also increased their levels of apoptosis. In addition, MIAT inhibition significantly impaired oxidized LDL (oxLDL) uptake of murine peritoneal as well as human monocyte-differentiated macrophages in vitro. In contrast, induction of MIAT expression by lipoprotein-a (LPa) treatment, displayed the opposite effect. Conditioned medium from macrophage cultures after MIAT knock-down substantially decreased hCASMC proliferation, indicating a potential involvement of MIAT in macrophage-SMC interactions during advanced stages of atherosclerosis.

Conclusion:
The lncRNA MIAT is a novel regulator of cellular processes in atherosclerosis and plaque stability, which influences SMC proliferation and apoptosis and interacts with disease-triggering macrophages.
Abstract 518: In Vitro and
in vivo
Disease Modeling Using Patient Derived iPSCs to Characterize the Calcification Phenotype in Arterial Calcification Due to Deficiency of CD73
Authors: St. Hilaire Cynthia , Jin Hui , Huang Yuting , Yang Dan , Negro Alejandra , Walts Avram , Liu Yangtengyu , Davaine Jean-Michel , Schwartzbeck Robin , Yu Zhen , Dmitrieva Natalia , Donahue Danielle , Chen Guibin , Boehm Manfred
Published: June 2021

Objective:
The objective of this study was to develop a patient-specific induced pluripotent stem cell (iPSC)-based disease model to understand the process by which CD73-deficiency leads to vascular calcification in the disease, Arterial Calcification due to Deficiency of CD73 (ACDC).

Approach & Results:
ACDC is an autosomal recessive disease resulting from mutations in the gene encoding for CD73, which converts extracellular AMP to adenosine. CD73-deficiency manifests with tortuosity and vascular calcification of the medial layer of lower-extremity arteries, a pathology associated with diabetes and chronic kidney disease. We previously identified that dermal fibroblasts isolated from ACDC patients calcify in vitro, however in vivo studies of the vasculature are limited, as murine models of CD73 deficiency do not recapitulate the human disease phenotype. Thus, we created iPSCs from ACDC patients and control fibroblasts. ACDC and Control iPSCs form teratomas when injected in immune-compromised mice, however ACDC iPSC teratomas exhibit extensive calcifications. Control and ACDC iPSCs were differentiated down the mesenchymal lineage (MSC) and while there was no difference in chondrogenesis and adipogenesis, ACDC iMSCs underwent osteogenesis sooner than control iPSC, have higher activity of tissue-nonspecific alkaline phosphatase (TNAP), and lower levels of extracellular adenosine. During osteogenic simulation, TNAP activity in ACDC cells significantly increased adenosine levels, however, not to levels needed for functional compensatory stimulation of the adenosine receptors. Inhibition of TNAP with levimisole ablates this increase in adenosine. Treatment with an A2b adenosine receptor (AR) agonist drastically reduced TNAP activity in vitro, and calcification in ACDC teratomas, as did treatment with etidronate, which is currently being tested in a clinical trial on ACDC patients.

Conclusions:
These results illustrate a pro-osteogenic phenotype in CD73-deficient cells whereby TNAP activity attempts to compensate for CD73 deficiency, but subsequently induces calcification that can be reversed by activation of the A2bAR. The iPSC teratoma model may be used to screen other potential therapeutics for calcification disorders.
Abstract 525: Cases with Uncommon Anti-phospholipid Antibody Syndrome Developed Multifocal Vascular Disorders Such as Ischemic Enteritis, Portal Vein Thrombosis, Ischemic Heart Disease and Cerebral Infarction
Authors: Kawamura Yasutaka , Nakaji So , Hirata Nobuto
Published: June 2021

Purpose:
We report cases with uncommon anti-phosholipid antibody syndrome developed ischemic enteritis, portal vein thrombosis, ischemic heart disease and cerebral infarction. The difficulty in diagnosing and treatment procedures for multifocal vascular disorders is discussed.

Patients and Methods:
Patient 1, 76-year-old man, with acute abdomen. Patient 2, 54-year-old man, with intractable esophageal varices. Clinical manifestation was superior mesenteric artery (SMA) thrombosis in the patient 1 and portal vein thrombosis in the patient 2. Serial CT scans were examined on 64 or 80 multidetector-row CT (Toshiba), with bolus injection of contrast media for CT angiography (CTA). Abdominal angiography with endovascular intervention and coronary angiography were performed on Allura Xper FD-20 and FD-10 (Philips).

Results:
Emergency CTA of patient 1 showed decreased contrast enhancement of SMA and SMV but enhancement of intestinal walls were well preserved. Although the patient’s symptom got better, follow-up CT revealed the growth of SMA thrombosis. The aspiration and the dissolution of SMA thrombosis were performed through the SMA catheter for five days resulted in complete recanalization.Then he felt chest oppression and 75% stenosis of RCA segment 4, followed by complete spastic occlusion on Acetylcholine loading on the coronary angiography, which recovered perfectly with intra-coronary nitroglycerin administration. He discharged our hospital with health, while he had cerebellar infarction with right cerebellar artery occlusion. On patient 2, to resolve portal hypertension, balloon-occluded retrograde transvenous obliteration was scheduled, however, due to numerous collaterals prevented the effective venous obliteration and splenic arterial embolization was selected alternatively.

Discussion and Conclusions:
The progressive SMA thrombosis with coronary disease and subsequent cerebellar occlusion seen in patient 1, and the portal vein thrombosis in patient 2 without chronic hepatitis nor cirrhosis were unusual. To discover underlining uncommon anti-phosholipid antibody syndrome is important to prevent serious vascular diseases in sequence.
Abstract 532: Protein Analysis of Tongxinluo-modulated Cytokines in Cardiac Microvascular Endothelial Cells via Tandem Mass Tag Quantitative Proteomics After Ischemia/Reperfusion Injury
Authors: Li Qing , Yang Yuejin
Published: June 2021

Background:
Proteomics is a potential tool to study the large-scale expression, function and interaction of the complement of proteins in an organism. In this study, we used the TMT-labeled proteomics to detect the various cytokines in an in vitro model of cardiac microvascular endothelial cells (CMECs) ischemia/reperfusion injury with Tongxinluo(TXL) treatment. Our aims are to investigate whether TXL could modulate the secretion function of CMECs, and to synthetically analysis the underlying mechanism of the regulation.

Methods:
Human CMECs were exposed to different concentrations of TXL, and incubated to scavenge free oxygen for 2 h of hypoxia and were moved to normal conditions for 2 h of reoxygenation. Cell apoptosis was assessed by flow cytometric analysis. CMECs were divided into three groups for TMT-labeled proteomics analysis: CMECs in normal condition (Group N), CMECs in hypoxia and serum deprivation condition (Group HR), CMECs treatment with TXL in hypoxia and serum condition(Group HR+TXL) . We utilized bioinformatics analysis to identify differential proteins.

Results:
TXL concentration-dependently decreased apoptosis of CMECs. The optimal concentration of TXL to have the maximum protection for CMECs was 800 μg/mL. Both hypoxia/reoxygenation and TXL treatment significantly changed the cytokines level of CMECs. 32 differential proteins between group N and group HR were detected. TXL treatment up-regulated 6 cytokines and down-regulated 6 cytokines in ischemia/reperfusion injury. These proteins mainly had vital functions such as cell proliferation, stress response, regulation of multicelluler organismal metabolic process. We evaluated several proteins played important roles in ischemia/reperfusion injury including Human Heme Oxygenase 1 (HMOX1), angiopoietin 2 (ANGPT2), sequestosome 1 (SQSTM1), and connective tissue growth factor (CTGF).

Conclusion:
The study presented differential proteins responsible for ischemia/reperfusion injury through TMT-labeled proteomic analysis. We assessed some vital proteins including their characters and roles. These findings may provide new mechanisms of TXL treatment in acute myocardial diseases.
Abstract 535: Trem Like Transcript-1 (TLT-1) Regulates Both Leukocytes and Endothelial Cells to Mediate Immunohemostasis
Authors: Washington A. V
Published: June 2021

TLT-1 is a 35kd receptor stored in the α-granules of platelets and released upon platelet activation like p-selectin. A soluble form is also released. A case has been made that the protein or proteins that regulate inflammatory bleeding is stored in the platelet α-granules. We hypothesized that TLT-1 is one of these proteins and we investigated TLT-1’s role in inflammatory bleeding. Using two different models of inflammation and three different models of transmigration we show that TLT-1 mediates neutrophil transmigration across the vessel wall. In the absence of TLT-1 transmigration leads to bleeding that can be partially rescued by soluble TLT-1 (sTLT-1: see poster J. Morales). Based on these results we hypothesized that TLT-1 enhances the progression of atherosclerosis and cancer. To test this hypothesis we crossed the

treml1
-/-

mice with apoe
-/-
mice creating the DKO mouse and evaluated lesion progression over 20 weeks. Our results demonstrate that the
DKO
has significantly less lesions in the aortic sinus than their littermate controls (n=8/group;
p
<0.05). The smaller lesions are associated with less platelet activation and fewer platelet-leukocyte conjugates. Parallel studies investigating TLT-1 in the α-granules revealed a significant co-localization with pro-angiogenic VEGF and higher sTLT-1 release with PAR1 vs PAR4 platelet activation; suggestive of a role for TLT-1 in angiogenesis. We subsequently asked if, “sTLT-1 has an effect on endothelial cells?” We show angiogenic effects of sTLT-1 on HUVECs that is blocked by the addition of anti-TLT-1, demonstrating it’s a sTLT-1 mediated process. Further studies using the Lewis lung carcinoma model demonstrate that the tumors in the null mouse have significantly fewer vessels than wildtype tumors and lower infiltration of macrophages (n=15). We subsequently show that sTLT-1 partially rescues the phenotype by increasing macrophage infiltration and increasing vessel growth (n=13) and that antibodies to TLT-1 recapitulate the

treml1
-/-

phenotype (n=10) suggesting anti-TLT-1 may be used as an intervention. We conclude that TLT-1 regulates both leukocytes and endothelial cells to mediate vascular integrity and is a plausible target for therapeutic interventions in cancer and atherosclerosis.
Abstract 540: Very Low Density Lipoprotein Assembly is Required for cAMP Responsive Element-binding Protein H Processing and Hepatic Apolipoprotein A-IV Expression in Mouse Models of Acute Steatosis
Authors: Cheng Dongmei , Xu Xu , Boudyguina Elena , Simon Trang , Deng Zhiyong , VerHague Melissa , Lord Caleb , Brown J. M , Lee Ann-Hwee , Weinberg Richard B , Parks John S
Published: June 2021

Background:
Our previous studies have demonstrated that hepatic expression of apolipoprotein A-IV (apoA-IV) is increased in mouse models of chronic steatosis and is closely correlated with hepatic triglyceride (TG) content. We have also shown that steatosis-induced hepatic apoA-IV gene expression is regulated by processing of the nuclear transcription factor cAMP responsive element-binding protein H (CREBH). Herein, we explored the mechanisms that mediate steatosis-induced CREBH processing.

Methods:
We measured hepatic CREBH processing, apoA-IV gene expression, and lipid content in several mouse models of attenuated or enhanced VLDL assembly that were subjected to acute steatosis induced by a 16 hour overnight fast or by feeding a ketogenic diet for 6 days.

Results:
Both fasting and the ketogenic diet induced acute hepatic TG accumulation associated with increased CREBH processing and apoA-IV gene expression, which were associated with hepatic TG content in C57BL/6 mice. All mouse models of attenuated VLDL secretion (shRNA-induced apoB knock down, liver specific microsomal triglyceride transfer protein (MTP) knockout, treatment with the MTP inhibitor BMS212122, and comparative gene identification-58 (CGI-58) deficiency) had increased hepatic TG accumulation, but displayed repressed CREBH processing and reduced apoA-IV gene expression compared to controls. When deficient VLDL assembly in liver-specific MTP knockout mice was reconstituted by adenoviral infection with a human MTP transgene, steatosis-induced CREBH processing and apoA-IV expression were restored. In a mouse model of enhanced VLDL assembly (transgenic overexpression of human MTP), apoA-IV gene expression correlated with bulk hepatic TG accumulation, but not with VLDL secretion rate, indicating that other steatosis-related factors participate in apoA-IV gene regulation.

Conclusions:
Taken together, these data provide compelling evidence that VLDL assembly and secretion is required for hepatic CREBH processing and enhanced apoA-IV gene expression in the setting of acute steatosis. These data further suggest that CREBH and apoA-IV play central roles in VLDL-mediated hepatic lipid efflux.
Abstract 542: Cellular Pip2 is Effluxed By Abca1 to Apoa1 and Pip2 Is Carried on Hdl That Can be Delivered to Target Tissues via Sr-b1
Authors: Gulshan Kailash , Brubaker Greg , Conger Heather , Wang Shuhui , Zhang Renliang , Hazen Stanley L , Smith Jonathan D
Published: June 2021

Introduction:
apoAI-ABCA1 mediated nascent HDL assembly promotes cholesterol efflux and reverse cholesterol transport. Cell surface phosphatidylinositol 4,5- bisphosphate (PIP2) mediates this process, but whether PIP2 is effluxed via apoA1-ABCA1 pathway and is carried in plasma is unknown.

Objective:
To determine if PIP2 is effluxed during ABCA1-dependent nHDL biogenesis, whether PIP2 is found on plasma HDL, and if PIP2 can be delivered to target cells via SRB1.

Methods and Results:
We previously reported at this meeting that: 1) apoAI binds to PIP2 with a K
d
of ~100 nM; 2) ABCA1 expression translocates PIP2 from the inner to outer leaflet of the plasma membrane; 3) cell surface PIP2 is required for ABCA1-dependent cellular binding of apoA1 and cholesterol efflux. Here, we report that PIP2 incubation with lipid-free apoA1 promoted monomeric vs. dimeric apoA1. We found that ABCA1 expression increased efflux of PIP2 by ~20-fold in RAW264.7 cells labeled with [3H]myo-inositol, and chased with apoA1 (mean ± SD; ***, p<0.001, two-tailed t-test, n=3). Plasma from apoA1 knockout (A1 KO), wild type (WT), and human apoA1 transgenic (A1-Tg) mice contained apoA1-gene dosage dependent levels of both cholesterol and PIP2, with 64-fold higher PIP2 levels in the A1-Tg vs. A1 KO mice (mean ± SD; **, p<0.01, two-tailed t-test, n=3). PIP2 was also reverse transported in vivo from [3H]myo-inositol-labeled macrophages implanted s.c into mice. Labeled PIP2 was recovered in the plasma, with a 4-fold higher of the injected radioactivity found in the WT vs. A1 KO host mice (mean ± SD; **, p<0.01, two-tailed t-test, n=3). FPLC separation of human plasma determined that almost all of the plasma PIP2 was found in the HDL fraction; and, human HDL contained two PIP2 species with either 18:0, 20:4 fatty acids or 16:0, 20:4 fatty acids, as detected by liquid chromatography tandem mass spectrometry. SR-BI-inducible BHK cells exhibited 2-fold higher uptake of [3H]PIP2-labeled HDL after SR-BI induction, indicating that HDL can deliver PIP2 to target cells (mean ± SD; **, p<0.01, two-tailed t-test, n=3).

Conclusions:
PIP2 is effluxed from cells via the apoA1-ABCA1 pathway and is carried on HDL. HDL can deliver PIP2 to target tissues via SR-B1.
Abstract 544: Strenuous Exercise and Quercetin Intake Differentially Modulate PCSK9 and ANGPLT4 in Normal C57BL6 Mice
Authors: Mahini Halleh , Alsayrafi Dhuha , Wu Tong , Garelnabi Mahdi
Published: June 2021

Introduction:
We previously reported that intake of quercetin during moderate exercise modulate lipid metabolism in LDLr -/- C57BL6 mice. The current study investigates the role of strenuous exercise and quercetin on lipids metabolism.

Study Design:
40 mice were divided into four groups (10 each). These groups are as follows: Control mice, left untreated; control quercetin group, orally supplied with 100 μg/day of quercetin without exercising; exercise group without quercetin, and exercise group with quercetin supplements. The exercise groups were run on a treadmill for 30 minutes, 20-30m/m/ 5 days/week for two month. All animals were on normal mouse chow, at the end of the two month treatment, tissues were collected and expressions of genes associated lipid metabolism were analyzed and the proteins Western Blotting were determined.

Results:
PCSK9 mRNA level was significantly up-regulated as the result of combination of exercise and quercetin intake (p< 0.05) or quercetin alone. ANGPLT3 mRNA level did not show any significant changes as a result of exercise or quercetin. However, ANGPLT4 mRNA levels significantly down-regulated with the combination after 8 weeks of exercise and quercetin intake compared to both control and Exercise (p < 0.05). ANGPLT4 also decreased with quercetin intake; however this change is not significant. There was a slight change in ANGPLT 4 levels in the exercise group.

Conclusion:
The combination of strenuous exercise and quercetin intake did not show any positive affect on LDL (plasma LDL levels were measured; however not presented above), this was also reflected by the upregulation of the PCSK9 gene expression. Lipoprotein related genes differentially modulated with the strenuous exercise and quercetin intake. This data suggest that the combination of strenuous exercise and quercetin intake unfavorably impact LDL associated PCSK9 gene; however differentially affect ANGPLT4 levels with the exercise or the combination.
Abstract 545: Differential mRNA Expression is Influenced by Apolipoprotein A-I in Order to Promote Foam Cell Regression
Authors: Maruko Elisa C , Xu Hao , Kaul Sushma , Capaldo Brian J , Pamir Nathalie , Lans Daniel S , Rich Stephen S , Thomas Michael J , Sorci-Thomas Mary
Published: June 2021

Atherosclerosis is a disease of both lipids and inflammatory immune cells. More specifically, elevated plasma levels of low-density lipoproteins (LDL) leads to migration of circulating monocytes into the artery wall. Lipid loaded monocyte cells subsequently proliferate in the arterial walls becoming macrophage foam cells; a hallmark of atherosclerotic lesions. A proposed mechanism of the protective effects of high-density lipoprotein (HDL) is apolipoprotein A-I (apo A-I) acting as a mediator of cholesterol efflux and subsequent foam cell regression. To better understand the biological changes stimulated by apo A-I treatment, differential expression analysis of microarray data was performed on spleen cells from apo A-I treated mice. LDL receptor null (LDLr
-/-
) and LDL receptor and apo A-I null (LDLr
-/-
, apoA-I
-/-
) mice were fed a western diet consisting of 0.2% cholesterol and 42% of calories as fat for 12 weeks. After 6 weeks of diet, a subset of mice for each genotype was subcutaneously injected with 200 micrograms of apo A-I 3 times a week for the remaining 6 weeks. The control group mice were subcutaneously injected with 200 micrograms of saline or BSA. Spleen cell RNA was isolated, purified, and analyzed for differential expression analysis using Illumina BeadArray Microarray Technology Analysis. Individual gene expression analysis for LDLr
-/-
, apoA-I
-/-
apo A-I treated mice showed 281 significantly differentially expressed genes compared to BSA treated mice. LDLr
-/-
A-I treated mice had 1502. Of the significant genes, 189 intersected across both genotypes. LDLr
-/-
, apoA-I
-/-
A-I mice showed 73 up-regulated and 116 down-regulated genes. Similarly, LDLr
-/-
A-I mice had 71 up-regulated and 118 down-regulated. One-directional Gene Set Enrichment Analysis (GSEA) of LDLr
-/-
, apoA-I
-/-
A-I mice revealed 49 significant pathways while a total of 63 were found for LDLr
-/-
. Of these pathways, 21 were up-regulated and 13 were down-regulated in both genotypes. Eight of the top 10 most significant up-regulated pathways in both genotypes were immune cell related. Their functions involve receptor, adhesion, and chemokine signaling. Overall, preliminary analysis suggests A-I treatment induces similar gene expression changes across different genotypes.
Abstract 546: Characterization of the I4399M Variant of Apolipoprotein(a): Implications for Altered Prothrombotic Properties of Lipoprotein(a)
Authors: McAiney Jackson , Scipione Corey , Simard Daniel , Gauld James , Boffa Michael , Koschinsky Marlys
Published: June 2021

Elevated plasma levels of lipoprotein(a) (Lp(a)) are a causal risk factor for CHD. Lp(a) closely resembles LDL, but contains an additional glycoprotein apolipoprotein(a) (apo(a)) that is structurally homologous to the fibrinolytic proenzyme plasminogen. Key sequence differences render the protease-like domain in apo(a) catalytically inactive. This has led to speculation that Lp(a) can oppose the fibrinolytic functions of plasminogen. A single nucleotide polymorphism (SNP) in the
LPA
gene encoding apo(a) results in an Ile to Met substitution at position 4399 in the protease-like domain. In population studies, this variant has been correlated with elevated plasma Lp(a) levels and with higher CHD risk, an effect that has been suggested to arise from effects of the variant on fibrin clot properties. We undertook a functional characterization of the effect of the I4399M substitution in apo(a). Molecular dynamics simulations of wild-type (wt) apo(a) and the Met variant revealed a shift from a buried (Ile) to slightly exposed (Met) environment, allowing for potential modification of the Met. Indeed, MALDI-TOF mass spectrometry analysis demonstrated the presence of a methionine sulfoxide moiety at this position in the Met variant. When 17-kringle recombinant forms of apo(a) were included in a plasma clot lysis assay, both the wt apo(a) and Met variant inhibited lysis, but the Met variant had a 50% greater effect. However, the Met variant was equally as efficient as wt apo(a) in inhibiting plasminogen activation on a fibrin surface. Apo(a) was also able to significantly shorten coagulation time for clots made from either purified fibrin or lipoprotein-deficient plasma, with the Met variant having twice as large an effect as wt apo(a). Morever, the Met variant resulted in greater turbidity of the clots whereas wt apo(a) had no effect. In agreement with these findings, SEM and confocal microscopy of fibrin clots showed that compared to wt apo(a), the I4399M variant resulted in significant alteration of the fibrin network, with a 4-fold increase in fibrin fiber width. Together, our data suggest that the Met4399 variant differs structurally from wt apo(a), which may underlie key differences related to its effects on fibrin clot architecture and fibrinolysis.
Abstract 547: Proteomic Correlation of Gel Filtration Lipoprotein Subfractions with Atheroprotective Functions of HDL
Authors: Swertfeger Debi , Li Hailong , Rebholz Sandra , Shah Amy S , Davidson W S , Lu L J
Published: June 2021

HDL has been shown to possess a variety of cardio-protective functions, including removal of excess cholesterol from the periphery, inhibition of oxidation, and stimulation of endothelial function. It has been proposed that various HDL sub-particles exist, each with distinct protein and lipid compositions. We hypothesized that fractionation of plasma would separate HDL particles with different protein compositions into distinct populations responsible for different HDL functions. Plasma from 10 healthy adults was fractionated by gel filtration and the protein composition of phospholipid containing fractions was analyzed by mass spectrometry. Each fraction was assessed for its ability to efflux cholesterol from macrophages, as well as its ability to inhibit LDL oxidation. Correlations were made between individual proteins in the HDL fractions and their ability to participate in both functional assays. One peak of activity was found in the cholesterol efflux assay when analyzing fractions in the HDL range. Cholesterol efflux activity did not correlate strongly with any protein in the HDL range of fractions, though there was a weak correlation with Protein S (r=0.337, p<0.01). However, the phospholipid (PL) and cholesterol (CH) concentration of the fractions correlated strongly with efflux (r=0.75. and r=0.61 respectively, both p<0.00001) across all fractions (both LDL and HDL). In contrast, the PL or CH content were not correlated with anti-oxidation activity. However, there were 2 strong peaks of anti-oxidation activity in the HDL range that did correlate with specific proteins (all values of r>0.3 and p<0.05). Activity in the first peak, fraction 25, in the small HDL range, correlated most strongly with ceruloplasmin and inter-α-trypsin inhibitor 4, while activity in the second peak, fraction 28, in the minimally lipidated/free protein range, correlated with gelsolin and albumin. In conclusion, we have shown that PL and CH correlate more strongly with cholesterol efflux than any protein in the HDL sub-fractions, while the protein composition of particular HDL sub-fractions is a better indicator of its anti-oxidative capacity than the PL or CH concentration.
Abstract 554: Mitochondrial Biomarkers for Assessing Early Tobacco Product Induced Cardiovascular Injury in Human Participants
Authors: Fetterman Jessica L , Holbrook Monica , Berk Brittany D , Weisbrod Robert M , Inagaki Elica , Linder Erika A , Breton-Romero Rosa , Keith Rachel , Bhatnagar Aruni , Hamburg Naomi M
Published: June 2021

Smoking is a major cause of preventable cardiovascular deaths worldwide. A number of new and emerging tobacco-related products including electronic(e)-cigarettes have become readily available as tobacco cigarette alternatives and are often marketed as being less harmful despite growing evidence that toxic aldehydes are absorbed into the circulation following use. However, there is insufficient scientific data to develop policy to regulate and evaluate the claims and toxicity of new tobacco-related products. Prior studies suggest that tobacco cigarette smoking induces cardiovascular oxidative stress which may be linked to mitochondrial abnormalities. Mitochondria are key cellular organelles that are sensitive to environmental toxins and are essential for maintaining cardiovascular health. Upon exposure to environmental stressors, mitochondria become damaged characterized by elevated oxidant production which drives further damage and oxidative stress. Hence, measures of mitochondrial function and damage may serve as novel biomarkers of tobacco product induced injury. We measured mitochondrial biomarkers as a detector of early toxicity of tobacco product-induced injury in peripheral blood mononuclear cells (PBMCs) from non-smoking controls, tobacco cigarette smokers, and e-cigarette users. Following antimycin A stimulation, mitochondrial oxidant production was elevated in PBMCs from both tobacco cigarette smokers and e-cigarette users compared to cells from non-smokers (8.5±0.6, 9.8±0.9 vs 7.0±0.4, p=0.03). Additionally, we assessed the mitochondrial oxidant production in vascular tissue from people utilizing novel techniques developed within our laboratory that allow us to study freshly isolated endothelial cells. Early preliminary data in freshly isolated venous endothelial cells from tobacco cigarette smokers suggests that mitochondrial oxidant production is elevated compared to cells from non-smokers (1.47±0.38 vs 0.96±0.1). This further suggests that mitochondrial biomarkers may have enhanced early sensitivity to tobacco product toxicity prior to overt clinical disease. Whether mitochondrial biomarkers relate to measures of vascular function will be the subject of future studies.
Abstract 561: Dysregulation of miR-155 in Acute Oscillatory Shear Stress (OSS)-mediated Oxidative Stress, Inflammation and Endothelial Dysfunction
Authors: Mohamed Islam , Thomas Sheena , Rooney Kimberly , Sutliff Roy , Willett Nick , Searles Charles
Published: June 2021

Introduction:
Shear stress forces play an integral role in dictating the endothelial cell (EC) response to changes in blood flow, pro-inflammatory response and hence development of atherosclerosis. Previously, our group has identified EC microRNA-155 (miR-155) as one of the key signature dysregulated miRNAs in areas of chronic low magnitude oscillatory shear stress (OSS) in vasculature and OSS models of in-vitro.

Hypothesis:
we hypothesized that acute induction of OSS mediates EC oxidative stress, inflammation and dysfunction, via dysregulation of EC miR-155.

Methods:
12-week old C57B/6J mice were subjected to abdominal aortic coarctation (AAC), a unique model of acute induction of OSS, for 3 days and downstream segments of acute OSS were compared to upstream unidirectional shear stress (USS) segments of the thoracic aorta.

Results:
Acute OSS resulted in down regulation of EC miR-155 expression and inverse upregulation of EC RhoA and Myosin light chain kinase (MYLK), known targets of miR-155-mediated EC cytoskeleton organization, in OSS segments compared with USS. This was associated with impaired EC dependent relaxation, differential contractile response to phenylephrine, and loss of EC barrier function as evaluated by extravasation of Evans-blue dye assay. In parallel, En-face immunohistochemical staining also showed increased expression of EC nitric oxide synthase (eNOS) along with increased levels of reactive oxygen species (ROS) and nitrotyrosine (NY) formation in OSS segments compared with USS.

Conclusions:
Together, our studies shed light on the early changes in EC response to acute induction of OSS and resulting down-regulation of EC mir-155, including; oxidative/inflammatory stress, EC dysfunction, loss of barrier function and cytoskeletal changes. Despite the early upregulation of eNOS, it could also potentially synergize with the activation of the RhoA-MYLK pathway in EC oxidative (ROS/NY)/inflammatory stress and associated EC dysfunction. Further studies are in progress to dissect the interplay between these different pathways and their causal relationships as downstream targets of EC miR-155.