bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 383: MLKL (Mixed Lineage Kinase Domain-like Protein) In The Splenic Microenvironment Restrains Hematopoietic Contributions To The Atherosclerotic Plaque
Authors: Rasheed Adil , Dennison Taylor , Wyatt Hailey , Nguyen My-anh , Robichaud Sabrina , Geoffrion Michele , Baxi Adir , Lee Richard , Ouimet Mireille I , Rayner Katey J
Published: November 2022

The mixed lineage kinase domain-like protein (MLKL) was first discovered in 2012 as the executioner of necroptosis. In line with this, we showed that MLKL knockdown (
Mlkl
KD) by administration of antisense oligonucleotides to atherosclerotic-prone
Apoe
-knockout (
Apoe
-/-) mice did indeed decrease necroptosis and necrotic core in the atherosclerotic plaque. However, there were no changes in overall plaque area, suggesting additional roles of MLKL during atherosclerosis. During atherogenesis, the spleen allows for the expansion of hematopoietic stem and progenitor cells (HSPCs) and mature myeloid cells that drive atherogenesis. Accordingly, we observed splenomegaly with an expansion of splenic HSPCs and mature myeloid cells upon
Mlkl
KD. However, bone marrow transplantation studies revealed that MLKL did not impact hematopoiesis through its expression in hematopoietic cells. Within hematopoietic reservoirs such as the spleen, HSPCs are regulated by their interaction with microenvironmental cell types, including endothelial cells. We thus
hypothesized
that MLKL preserves the microenvironment to repress hematopoiesis in the spleen during atherosclerosis. Flow cytometry and immunofluorescence staining during a time course of atherosclerosis revealed a progressive decrease of splenic endothelial cells that was correlated with a reduction of MLKL endogenously or by
Mlkl
KD, suggesting that splenic MLKL modulates the endothelial cells known to regulate hematopoiesis. RNA sequencing of these splenic endothelial cells revealed two major pathways that were perturbed upon
Mlkl
KD, including cell cycle and factors related to leukocyte differentiation. Adoptive transfer of CD45.1 HSPCs to CD45.2
Apoe
-/- mice during
Mlkl
KD revealed an expansion of mature immune cells in the spleen as compared to control, together demonstrating the influence of MLKL on the splenic microenvironment and subsequently hematopoiesis. In conclusion, we here demonstrate a novel role for MLKL in regulating the balance of hematopoiesis, specifically through preservation of splenic endothelial cells that promote HSPC quiescence, and more generally highlight the contributions of the spleen and its impact on inflammatory pathways that potentiate atherogenesis.
Abstract 385: Fortilin Interacts With Tgf-β1 And Prevents Tgf-β Receptor Activation
Authors: Pinkaew Decha , Martinez-Hackert Erik , Jia Wei , King Matthew , Miao Fei , Enger Nicole , Silakit Runglawan , Ramana Kota , Chen Shiyou , Fujise Ken
Published: November 2022

Fortilin is a 172-amino acid multifunctional protein present in both intra- and extracellular spaces. Although fortilin binds and regulates various cellular proteins, the biological role of extracellular fortilin remains unknown. Here we report that fortilin specifically interacts with TGF-β1 and prevents it from activating the TGF-β1 signaling pathway. In a standard immunoprecipitation-western blot assay, fortilin co-immunoprecipitates TGF-β1 and its isoforms. The modified ELISA assay shows that TGF-β1 remains complexed with fortilin in human serum. Both bio-layer interferometry and surface plasmon resonance (SPR) reveal that fortilin directly bind TGF-β1. The SPR analysis also reveals that fortilin and the TGF-β receptor II (TGFβRII) compete for TGF-β1. Both luciferase and secreted alkaline phosphatase (SEAP) reporter assays show that fortilin prevents TGF-β1 from activating Smad3 binding to Smad-binding element (Figure). Fortilin inhibits the phosphorylation of Smad3 in both quantitative western blot assays and ELISA. Finally, fortilin inhibits TGFβ-1-induced differentiation of C3H10T1/2 mesenchymal progenitor cells to smooth muscle cells. A computer-assisted virtual docking reveals that fortilin occupies the pocket of TGF-β1 that is normally occupied by TGFβRII and that TGF-β1 can bind either fortilin or TGFβRII at any given time. These data support the role of extracellular fortilin as a negative regulator of the TGF-β1 signaling pathway.
Abstract 420: Proprotein Convertase Subtilisin/Kexin 6 Is Involved In Lipid Metabolism In Liver
Authors: Suur Bianca , Chemaly Melody , Karadimou Glykeria , Jin Hong , Kronqvist Malin , Lengquist Mariette , van der Laan Sander W , Sabater Lleal Maria , Mälarstig Anders , Gerard Pasterkamp , Eriksson Per , Hedin Ulf , Hurt-Camejo Eva , Ketelhuth Daniel F , Matic Ljubica
Published: November 2022

Introduction:
PCSK6 is a protease that activates cytokines and growth factors and strongly enriched in healthy human liver, however its function in this context has not been explored. We have previously shown that PCSK6 is induced in atherosclerotic plaques from patients with symptoms of stroke and important for regulating several cell types in this context. Here, we aimed to investigate the role of PCSK6 in lipid metabolism in liver, particularly in the context of atherosclerosis and non-alcoholic fatty liver disease (NAFLD).

Methods:
We used publically available datasets and several atherosclerosis biobanks to investigate the expression of PCSK6 in healthy and diseased human tissues. In addition, we used full
Pcsk6
-/-
mice as well as liver specific conditional
Pcsk6
-/-
knockout mice compared to littermate controls, to investigate the effects of PCSK6 ablation on lipid metabolism.

Results:
Genetic analyses of the PCSK6 locus identified a variant, rs7181043, that was significantly associated with PCSK6 mRNA expression in healthy human adipose tissue, liver and in atherosclerotic plaques. The same variant was associated specifically with plaque fat content and atherosclerotic patient’s plasma LDL levels. In addition, PCSK6 mRNA expression in plaques was positively correlated with total plasma cholesterol and LDL levels in atherosclerotic patients as well as lipid metabolism associated pathways within the carotid plaque. Microarray comparison of the livers from
Pcsk6
-/-
mice and controls showed that VLDL particle assembly was one of the upregulated processes.
I
n vivo
studies showed that
Pcsk6
-/-
mice have higher plasma cholesterol and LPL levels at baseline compared to controls, and lower levels of LDLR in their liver. These findings were further confirmed in liver specific conditional knockouts. Preliminary results show that liver specific knockout mice develop increased liver steatosis and fibrosis on a modified western diet.

Conclusions:
Our data suggests that PCSK6 is involved in cholesterol and metabolic control in liver. Breeding of liver specific Pcsk6 knockout mice on an ApoE
-/-
background is currently ongoing and will provide insight into the role of liver PCSK6 in atherosclerosis and NAFLD development.
Abstract 425: Genetic Inhibition Of Hepatic Dolichol Biosynthesis Results In Hypolipidemia
Authors: De Giorgi Marco , Doerfler Alexandria M , Hurley Ayrea , Walkey Christopher , Lagor William R
Published: November 2022

Dolichol is a nonsterol isoprenoid derived from the mevalonate pathway. Dehydrodolichyl diphosphate synthase subunit (DHDDS) is a branch point enzyme that - in complex with Nogo-B receptor (NgBR) - catalyzes the first committed step of dolichol biosynthesis. Next, dolichol is phosphorylated, and used as carrier for oligosaccharides, which are transferred to nascent proteins in the N-linked glycosylation pathway. A growing body of evidence highlights the importance of N-linked glycosylation in regulating plasma lipid levels in humans. However, the underlying molecular mechanisms are largely unexplored. Therefore, the goal of this project is to define the role of dolichol biosynthesis in lipid metabolism in the liver, the major organ for the biosynthesis and clearance of lipoproteins. We used Adeno-Associated Virus (AAV)-mediated delivery of CRISPR/Cas9 (AAV-CRISPR) to delete
Dhdds
in mouse liver, to inhibit the hepatic biosynthesis of dolichol. Mice were injected with AAV-CRISPR or saline as control, and fed a high-fat diet for 4 to 12 weeks. Surprisingly,
Dhdds
liver-specific knockout (LSKO) mice gained less weight compared to control mice. Plasma total cholesterol and triglyceride levels were also reduced over time in
Dhdds
LSKO mice. Moreover, we observed less cholesterol content in the high-density lipoprotein (HDL) fractions as well as less triglycerides in very-low-density lipoproteins (VLDL). At the cellular level,
Dhdds
LSKO mice showed hepatic endoplasmic reticulum (ER) stress and decreased N-linked glycosylation. In addition, we observed significant downregulation of Sterol Regulatory Element-Binding Protein 1c (
Srebp-1c
) - a key regulator of
de novo
lipogenesis (DNL) - and downstream-regulated genes in the DNL pathway. Conversely, the expression of genes regulating cholesterol biosynthesis was unaffected. Taken together, our data show an important role for hepatic dolichol biosynthesis in regulating plasma lipids, potentially through modulation of
Srebp-1c
expression and DNL.
Abstract 433: Investigation Of The Divergent Roles Of Macrophage Mtor Signaling In Atherosclerosis
Authors: Zhang Xiangyu , Evans Trent , Sunny Chen , Sergin Ismail , Jeong Se-jin , Rodriguez-Velez Astrid , Yeh Yu-sheng , Stitham Jeremiah , Park Arick , Razani Babak
Published: November 2022

The mechanistic target of rapamycin (mTOR) pathway is considered a key regulator of cellular proliferation, survival, metabolism, and degradation. Yet its role in atherosclerosis and in macrophages of the atherosclerotic plaque remains poorly understood. Our previous study has demonstrated macrophage mTORC1 signaling promotes atherogenesis through inhibiting autophagy and increasing apoptosis. Using macrophage-specific Rictor- and mTOR-deficient mice, we now dissect the distinct functions of mTORC2 pathways in atherogenesis. In contrast to the protective effect against atherosclerosis from blocking macrophage mTORC1, macrophage-specific mTORC2-deficient mice have the opposite phenotype with the development of larger and more complex lesions. In cultured macrophages, we show that mTORC2 signaling inhibits the FoxO1 transcription factor leading to suppression of pro-inflammatory pathways, especially the inflammasome/IL-1β response, which also manifest in vivo. In addition, administration of FoxO1 inhibitors efficiently rescued the hyperinflammation caused by mTORC2 deficiency in vitro and in vivo. Interestingly, deletion of macrophage mTOR, which ablates both mTOR-dependent pathways leads to minimal change in plaques reflecting the apparently balanced and opposing roles of its signaling arms. Our data provide the first mechanistic details of macrophage mTOR signaling in atherosclerosis and suggest that therapeutic measures aimed at modulating mTOR need to account for its dichotomous functions.
Abstract 445: Pulmonary Surfactant Protein B Carried By HDL Is Associated With Incident Cardiovascular Disease In Patients With Type 1 Diabetes
Authors: Shao Baohai , Snell-bergeon Janet , De Boer Ian H , Kothari Vishal M , Davidson Sean S , Bornfeldt Karin E
Published: November 2022

Atherosclerotic cardiovascular disease (CVD) is the major cause of death in people with type 1 diabetes mellitus (T1DM). Alterations in the HDL proteome associate with prevalent CVD in T1DM. We therefore determined which proteins carried by HDL are associated with incident CVD in T1DM patients. We conducted a case-cohort study of incident CVD in the prospective Coronary Artery Calcification in Type 1 Diabetes (CACTI) study. We randomly sampled 145 CACTI participants without prevalent CVD, 11 of whom subsequently developed a CVD event as a cohort, plus all additional T1DM participants in CACTI (N=36) who developed an incident CVD event (a total of 47 CVD cases). Using targeted MS/MS, we quantified 51 proteins in HDL isolated from baseline plasma samples. We used Cox proportional regression analysis and a case-cohort design to test associations of HDL proteins with incident CVD (myocardial infarction, coronary artery bypass grafting, angioplasty, or death from coronary heart disease). Only one HDL protein—SFTPB (pulmonary surfactant protein B)—was associated with incident CVD in T1DM in all of the models tested. In a fully adjusted model that controlled for smoking status, lipids and other risk factors, the hazard ratio was 2.17 per SD increase of SFTPB (95% confidence interval, 1.12-4.21, P=0.022). Plasma fractionation and targeted MS/MS quantification demonstrated that nearly 95% of plasma SFTPB is quantitatively bound to HDL. Our results show that elevated levels of SFTPB in HDL are strongly associated with incident CVD in CACTI subjects with T1DM independently of smoking status and a wide range of other confounding factors. Because HDL is the major carrier of SFTPB in plasma, our observations support the proposal that SFTPB carried by HDL is a novel marker of CVD risk in patients with T1DM.
Abstract 446: Evaluation Of Zbp1 As An Atherosclerosis Modifier Gene
Authors: Smith Jonathan D , Han Juying
Published: November 2022

Objective:
To identify and validate the candidate gene responsible for the mouse strain effect on atherosclerosis in
Apoe
-/-
mice in the
Ath28.1
Quantitative Trait Locus (QTL) on the distal end of chromosome 2.

Background:
Our prior
Apoe
-/-
AKR x DBA/2 mouse strain intercross identified the strongest locus associated with aortic root lesion area on chromosome 2, called the
Ath28
QTL. Congenic mice identified three different atherosclerosis modifying subregions. The
Ath28.1
QTL subregion is the smallest and contains only three protein coding genes.

Methods:
Bioinformatic analysis of the coding and non-coding gene differences in the
Ath28.1
QTL between the AKR and DBA/2 strains was assessed using the Sanger Mouse Genomes Project. Bone marrow derived macrophages (BMDM) from DBA/2 and AKR mice were cultured for RNAseq analysis of expression of genes in the
Ath28
QTL region.
Zbp1
knockout mice have been obtained for atherosclerosis assessment.

Results and Conclusion:
There are only three protein coding genes in the
Ath28.1
QTL interval,
Pck1, Zbp1
, and
Pmepa1
. Sequence analysis identified one missense variant and one 3’UTR variant between the
Zbp1
genes in the AKR and DBA/2 mouse strains. The
Zbp1
gene encodes the Z-DNA binding protein 1, also called DAI, the DNA-dependent activator of interferon-regulatory factors.
Zbp1
plays a role in necroptosis and inflammation via its actions in the RIPK1, RIPK3, and MLKL pathways. BMDM from both strains were subjected to RNAseq analysis revealing higher expression of
Zbp1
in the DBA/2 strain. In addition, RNAseq showed expression of a long and a short RNA/protein isoforms of
Zbp1
, due to alternative splicing. The DBA/2 strain had ~ equal number of splice junction reads for the long and short isoforms, while the AKR strain had ~3-fold higher number of splice junction reads for the short vs. the long isoform. Fisher’s exact test showed this splice usage difference was significant (p=0.048). Ongoing
Zbp1
functional studies in stem cell derived macrophages will determine if the missense variant or splice differences alter Zbp1 activity in necroptosis.
Zbp1
KO mice are being treated with LDLr antisense oligonucleotides to determine if
Zbp1
plays a role as an atherosclerosis modifier gene.
Abstract 449: Post-transcriptionally Modified TRNA-derived Small RNA On HDL Antagonize Atherosclerosis Inflammation
Authors: Semler Elizabeth M , Michell Danielle L , Allen Ryan M , Ramirez-Solano Marisol , Linton MacRae F , Sheng Quanhu , Vickers Kasey C
Published: November 2022

Atherosclerotic cardiovascular disease (ASCVD) is a complex disease of uncontrolled hypercholesterolemia and inflammation. High-density lipoproteins (HDL) have been shown to suppress atherogenesis through multiple processes; however, many of HDL’s beneficial properties are lost during ASCVD. While the underlying reasons for the switch are unknown, changes to the composition and chemical properties of HDL cargo are likely a primary contributing factor. We have previously reported that HDL transfer microRNAs to recipient cells where they regulate gene expression. Based on our small RNA (sRNA) sequencing data, HDL also transports multiple classes of host and non-host sRNAs, including sRNAs-derived from parent tRNAs (tDRs). Recent studies indicate that extracellular sRNAs are post-transcriptionally modified and provide an additional layer of gene regulation. The current barrier to studying HDL-sRNA modifications is that modified sRNAs are often not captured by conventional sRNA sequencing approaches. To overcome this barrier, we have performed AlkB-facilitated RNA Methylation sequencing which removes common modifications on sRNAs prior to sequencing that normally would interfere with reverse transcription. Preliminary results support that HDL-tDRs are indeed heavily modified and conventional sequencing approaches greatly underestimate extracellular HDL-sRNA abundances. Previous work has established that modified sRNA antagonizes inflammation, primarily through inhibition of endosomal RNA-sensing toll-like receptors (i.e., TLR8) within immune cells. On the contrary, other studies have demonstrated that unmodified extracellular sRNAs can serve as TLR8 agonists. Due to the observations that
a)
HDL is highly-enriched in tDRs,
b)
HDL-tDRs are post-transcriptionally modified,
c)
modified sRNAs inhibit TLR signaling and inflammation, we hypothesize that modifications to HDL-tDRs confer antagonism towards immune cell activation and atherosclerosis inflammation.
Abstract 451: Enrichment Of High Density Lipoprotein-Associated Protease Inhibitor Activity Prevents Atherosclerosis Progression
Authors: Mobilia Maura , Whitus Callie , Karakashian Alexander , Gordon Scott M
Published: November 2022

Background:
Atherosclerotic lesions contain inflammatory cells which secrete a variety of proteolytic enzymes that contribute to lesion progression. The relative contributions of specific proteases to lesion progression is not well understood. The serine protease elastase is produced by neutrophils and macrophages in atherosclerotic lesions and may contribute to plaque progression and destabilization. We have reported significant enrichment of protease inhibitor proteins associated with plasma high density lipoprotein (HDL), including the elastase inhibitor alpha-1-antitrypsin, suggesting a potential role for HDL as an endogenous modulator of protease activity.

Hypothesis:
We hypothesized that protease inhibition is a mechanism for HDL-mediated protection against atherosclerosis that is independent of HDL cholesterol content.

Methods and Results:
We developed an HDL-targeting protease inhibitor (HTPI) peptide to allow for synthetic enrichment of HDL-associated elastase inhibitor activity. The small (1 kDa) peptide has an HDL-targeting domain, a soluble linker, and an elastase inhibitor motif. Using a fluorescent version, we demonstrated preferential binding to HDL in human plasma
ex vivo
. When injected into mice, we observed binding to plasma HDL and increased elastase inhibitor activity on HDL isolated from plasma of mice that received HTPI. Injection into mouse models of atherosclerosis resulted in accumulation of HTPI within lesions. To examine the impact of HTPI treatment on established atherosclerosis,

Ldlr
-/-

mice were maintained on Western diet for 12 weeks, then administered vehicle or HTPI (2.5 mg/kg) three times per week for two weeks while remaining on diet. Lesion quantification by
en face
analysis and aortic root sections demonstrated that HTPI prevented additional lipid deposition in established lesions. The impact of HTPI on lesion morphology and inflammatory features was examined by histology and immunofluorescence staining of aortic root sections.

Conclusions:
Targeted enrichment of HDL-associated elastase inhibitor activity using a synthetic peptide prevented atherosclerotic lesion progression. These findings support a possible role for the endogenous antiprotease activity of HDL in atheroprotection.
Abstract 487: Extracellularhistonestrigger Disseminated Intravascular Coagulation By Lytic Cell Death
Authors: Wu Congqing , Zhang Yan , Li Lan , Pandeya Ankit , Zhang Guoying , Cui Jian , Kirchhofer Daniel , Wood Jeremy P , Smyth Susan S , Wei Yinan , Li Zhenyu
Published: November 2022

Background:
Histones are cationic nuclear proteins that are essential for the structure andfunctions of eukaryotic chromatin. However, extracellular histones trigger inflammatoryresponses and contribute to death in sepsis by unknown mechanisms. We recently reported thatinflammasome activation and pyroptosis trigger coagulation activation through a tissue factor(TF)-dependent mechanism.

Objective and methods:
We used a combination of various deficient mice to elucidate the molec-ular mechanism of histone-induced coagulation.

Results:
We show that histones trigger coagulation activation
in vivo
as evidenced bycoagulation parameters and fibrin deposition in tissues. However, histone-induced coagulopathywas neither dependent on intracelluar inflammasome pathwyas involving caspase 1/11 andgasdermin D (GSDMD), nor on cell surface TLR2 and TLR4 mediated host immune response, asdeficiency of these genes in mice did not protect against histone-induced coagulopathy.Incubation of histones with macrophages induced lytic cell death and phosphatidylserine (PS)exposure, which is required for TF activity, a key initiator of coagulation. Neutralization of TFdiminished histone-induced coagulation.

Conclusion:
Our findings reveal lytic cell death as a novel mechanism of histone-inducecoagulation activation and thrombosis.
Abstract 489: Glutathione Regulates Endothelial Cell Function And Vegfr2 Signaling
Authors: Rashdan Nabil A , Mackay Ryan , Delgadillo Luisa , Pattillo Christopher B
Published: November 2022

Under physiological and pathological conditions angiogenesis progresses in an idealized redox window. Mice heterozygous for the Glutamate cysteine ligase modifier (GCLm) subunit knockout have augmented ischemic remodeling. During occlusion, the ensuing ischemic milieu and accompanying vascular changes regulate the activity of vascular endothelial receptor 2 (VEGFR2). We hypothesized that the cellular GSH concentration regulates endothelial cell function via altered VEGFR2 signaling. To test this hypothesis, we first performed mouse aortic endothelial cell (MAEC) outgrowth assays on aortic segments dissected from mice homozygous (KO) and heterozygous (Het) for GCLm knockout as well as wildtype control (WT). Outgrowth area in the Het group was significantly higher than WT and KO at day 7 of outgrowth (2.15 fold, n=4, P<0.001). MAECs were isolated and relative migration rates were determined using under-agarose migration assays. Het MAECs were 1.25 fold faster than WT (n=4 p<0.001) while KO MAECs were slower, 0.90 fold (n=3, p<0.05). Cell proliferation assays are currently underway. Concurrently, we inhibited glutathione reductase in primary human aortic endothelial cells using 2-AAPA. 2-AAPA (75 μm) alone didn’t activate VEGFR2 (determined by western blot for pY1175 VEGFR2). 2-AAPA in combination with H
2
O
2
(100 μM) induced a 5.5 fold (p<0.01, n=4) increase in the relative amount of active VEGFR2 compared to vehicle treated HAECs. 2-AAPA with H
2
O
2
induced a significant increase in pY416 proto-oncogene c-Src (Src) 9.5 fold (p<0.01, n=6), This effect was attenuated by the addition of DTT. Furthermore, Src kinase inhibitor 1 (SKI) (200 nM) attenuated the effect of 2-AAPA + H
2
O
2
on VEGFR2. While the VEGFR2 kinase inhibitor SU1948 inhibited activation of VEGFR2, it had no significant effect on the 2-AAPA + H
2
O
2
mediated activation of Src. Combined these data suggest Src mediates the 2-AAPA + H
2
O
2
activation of VEGFR2. Glutathione S-transferase P1 (GSTP1) catalyzes the enzymatic glutathionylation of proteins, The GSTP1 inhibitor TLK199 (50 μM) abrogated the effects of 2-AAPA + H
2
O
2
on VEGFR2 and experiments are underway to determine the glutathionylation status of Src in our system and its effect on the interaction of Src and VEGFR2.
Abstract 495: Effect Of Lipid Composition On The Atheroprotective Properties Of High-Density Lipoprotein-Mimicking Micelles
Authors: Hong Kristen W , Yu Minzhi , Mei Ling , Olsen Karl , Guo Yanhong , Luo Yonghong , Chen Eugene , Schwendeman Anna
Published: November 2022

An imbalance of cholesterol metabolism and unresolved local inflammation on the arterial wall drives atherosclerosis progression. Synthetic high-density lipoproteins (sHDL) have been shown to promote reverse cholesterol transport and reduce vascular inflammation, but producing sHDL at a large scale is challenging due to the difficulty in manufacturing recombinant Apolipoprotein A-I. HDL-mimicking micelles have been designed to mimic the structure and function of HDL. Prior research has shown that the phospholipid (PL) composition of HDL affects its overall activity, thus we have examined how PL composition of micelles affects it’s anti-inflammatory properties and cholesterol mobilization capacity. Micelles were composed four phosphatylcolines (PC), either POPC, DMPC, DPPC or DSPC, and DSPE-PEG2k. A second library of micelles was created using different ratios of DMPC and DSPE-PEG. Micelles ranged from 14 to 28 nm in diameter in size.
In vitro
, POPC and DMPC micelles were able to reduce TNF-α and IL-6 cytokine levels, diminish activation of nuclear factor-κB (NF-κB) and dissolve cholesterol crystals to a greater extent than DPPC and DSPC micelles. 1:2 DMPC:DSPE-PEG micelles caused the greatest percentage of cholesterol effuxed from cells (~8%) .
In vivo
PK/PD results showed that the PC composition does not greatly affect the PK or cholesterol mobilization of micelles. On the contrary, micelles with a 1:3 DMPC:DSPE-PEG ratio had a larger AUC (22815.2 mg*h/dL) and C
max
(775.0 mg/dL) when compared to the 1:0.5 ratio micelles (14266.1 mg*h/dL and 523.2 mg/dL, respectively). The maximum amount of cholesterol mobilized over 48 hours was also greater in the 1:3 ratio vs 1:0.5 ratio micelles (7813.3 mg*h/dL vs 3883.3 mg*h/dL). In conclusion, the composition of HDL-mimicking micelles affects it’s
in vitro
anti-atherosclerotic properties, such as reduction of pro-inflammatory cytokine release, diminished activation of NF-kB, cholesterol crystal dissolution and cholesterol efflux capabilities, as well as cholesterol mobilization
in vivo
. Further investigation must be completed in order to fully understand how lipid properties influence the therapeutic potential of micelles to treat atherosclerosis.
Abstract 496: The Cardiometabolic Disease Risk Protein SVEP1 Activates AKT/mTOR By Signaling Through The Orphan Receptor PEAR1
Authors: Elenbaas Jared S , Pudupakkam Upasana , Patel Ved , Ashworth Katrina , Kang Chul Joo , Jung In-Hyuk , Santana Katherine , Alisio Arturo , Di Paola Jorge , Stitziel Nathan
Published: November 2022

Background:
Sushi, von Willebrand factor type A, EGF and pentraxin domain containing 1 (SVEP1) is an extracellular matrix protein that circulates in plasma and is causally related to cardiovascular disease, hypertension, and type 2 diabetes. A recent genome wide association study (GWAS) also implicates
SVEP1
in platelet reactivity. The gene most strongly associated with platelet reactivity in the GWAS is
Platelet and Endothelial Cell Receptor 1
(
PEAR1
), a gene that encodes an orphan receptor tyrosine kinase-like protein that also associates with cardiovascular disease. Little is known about the molecular interactions and disease mechanisms of these proteins, despite their associations with cardiovascular disease and platelet reactivity.

Methods:
We used Mendelian Randomization (MR) and phenome-wide association studies to identify candidate SVEP1 protein interactions. We tested candidate interactions using recombinant proteins with molecular, cellular, and ex vivo assays.

Results:
A coding variant within the ectodomain of PEAR1 alters plasma levels of SVEP1 in humans (p=1.7x10^-17), suggesting these proteins may interact. MR demonstrates that genetically determined, increased plasma PEAR1 levels causally associate with decreased plasma SVEP1 (p=1.3x10^-11), further supporting an interaction. PEAR1 co-immunoprecipitates with SVEP1. Exposure to SVEP1 induces PEAR1 phosphorylation and activation of AKT/mTOR signaling in endothelial cells, vascular smooth muscle cells, and platelets. siRNA knockdown of PEAR1 abrogates this signaling. Endothelial cells adhere and proliferate in response to immobilized SVEP1. Platelets isolated from mice lacking SVEP1 have lower activation relative to controls and phenocopy platelets from mice lacking PEAR1.

Conclusions:
Despite robust evidence for the role of SVEP1 and PEAR1 in cardiovascular disease, critical gaps remain in our understanding of their disease mechanisms. Here, we identify SVEP1 as the first biological ligand of PEAR1 and a potent activator of AKT/mTOR signaling. The interaction between SVEP1 and PEAR1 is particularly intriguing, given the prominent role of AKT/mTOR in cardiovascular disease, and disrupting the interaction may be therapeutically beneficial.
Abstract 505: Cross Talk Between T Cells And Vascular Smooth Muscle Cells May Contribute To Phenotypic Changes In Human Coronary Plaque
Authors: Veizades Stefan , Jensen Annie , Tso Alexandria , Huang Xianxi , D'addabbo Jessica , Chien Yueh-hsiu , Roy Chowdhury Roshni , Chan Charles , Davis Mark , Nguyen Patricia
Published: November 2022

Background:
The atherosclerotic plaque is a complex niche composed of immune cells including T cells and macrophages as well as vascular smooth muscle cells. How T cells interact with the plaque microenvironment is not well understood.

Objective:
To map the crosstalk between T cells, macrophages, and smooth muscle cells.

Methods/Results:
We performed single cell RNAseq using the 10x transcriptomics platform in coronary plaque samples from 12 patients, who underwent heart transplantation. We found a high proportion of macrophages as well as T cells, many of which display a memory phenotype that was confirmed by immunohistochemistry. Ligand-receptor computational analysis demonstrated significant cross talk between T cells, macrophages, and smooth muscle cells. One cytokine of interest is amphiregulin (AREG), which has been shown to mediate tissue healing and fibrosis. Of note, AREG expression appears to increase as coronary plaques progress from early lipid-rich stages to more advanced stages. To elucidate the effects of AREG on smooth muscle cells, we treated human coronary artery smooth muscle cells (hCASMCs) in vitro with recombinant AREG and found increased cell proliferation compared to control (P<0.001). Bulk RNAseq profiling of hCASMCs showed activation of pathways that promote inflammation, proliferation, and fibrosis (Figure 1).

Conclusion:
Taken together, our findings suggest that cross talk between T cells and smooth muscle cells, partially through AREG production by T cells, may contribute to plaque progression.
Abstract 518: Erk5 S496 Phosphorylation, But Not Erk5 Kinase Or Transcriptional Activity, Is Responsible For Promoting Macrophage Inflammation And Mitochondrial Dysfunction Via Upregulating Novel Site Of Nrf2 K518 Sumoylation
Authors: Kotla Sivareddy , Imanishi Masaki , Zhang Aijun , Ko Kyung Ae , Samanthapudi Venkatasubrahman , Savage Hannah , Schadler Keri , Abe Rei , Deswal Anita M , Lin Steven , Reyes-Gibby Cielito , Yeung Sai-Ching , Pownall Henry J , Fujiwara Keigi , Hamilton Dale , Li Shengyu , Wang Guangyu , Le Nhat-Tu , Abe Jun-ichi
Published: November 2022

ERK5 is a dual kinase-transcription factor, which contains two transcriptional transactivation domains in the C-terminus and a kinase domain in the N-terminus. Many ERK5 kinase inhibitors have been developed, and are being tested in clinical studies for cancer and inflammatory diseases. Recent data has raised questions regarding the functional role of these ERK5 kinase inhibitors. Specifically, the possible link between blockade of pro-inflammatory ERK5 S496 phosphorylation and the anti-inflammatory effects of ERK5-specific kinase inhibitors has largely been neglected. In this study, we aimed to study the role and regulatory mechanisms of ERK5 S496 phosphorylation on macrophage inflammation and the impact of ERK5-specific kinase inhibitors. ATP binding site deletion mutant of ERK5b (a kinase-dead mutant) inhibited KLF2 induction but not oxidized LDL (oxLDL)-induced ERK5 S496 phosphorylation and TNFα expression. In contrast, both specific ERK5 kinase inhibitors (AX15836 and XMD8-92) and a dual phosphorylation site mutant of ERK5 (AEF) inhibited not only KLF2 but also oxLDL-induced ERK5 S496 phosphorylation and TNFα induction. These data suggested that ERK5 S496 phosphorylation, but not ERK5 kinase activity, plays a crucial role in ERK5-mediated pro-inflammatory effects. We also discovered a key effect of ERK5 S496 phosphorylation on SUMOylation at a novel site of NRF2 (i.e., K518), which inhibited NRF2 transcriptional activity without affecting ERK5 kinase activity, and antagonized oxLDL-induced macrophage inflammation. The role of NRF2 activation on the efficiency of oxidative phosphorylation (OXPHOS) and ATP synthesis had previously been reported, and we found that both ERK5 S496A and NRF2 K518R mutants abolished oxLDL-induced reduction of OXPHOS, ATP, and NAD
+
levels. In summary, we discovered a novel mechanism in which ERK5 S496 phosphorylation directly inhibited NRF2 activity via SUMOylation of NRF2 at K518 and thereby induced macrophage inflammation and mitochondrial dysfunction. The often-neglected role of ERK S496 signaling should be carefully considered in the interpretation of prior reports of ERK5 knockdown and pharmacological kinase inhibition relative to cellular inflammation and mitochondrial dysfunction.
Abstract 522: Macrophage Galectin 3 Protects Against Atherosclerosis By Promoting Repair, Removal And Replacement Of Damaged Lysosomes
Authors: Rodirguez-Velez Astrid C , Razani Babak , Zhang Xiangyu , Jeong Se-jin
Published: November 2022

Understanding the derangements in cellular processes that underlie macrophage dysfunction is critical to the development of future therapeutics. The autophagy-lysosome system in macrophages plays critical role in the degradation and recycling of long-lived/damaged intracellular material. Atherosclerotic plaque macrophages develop an impairment in autophagy and lysosomes which is a contributing factor to plaque progression. Thus, one can surmise that harnessing programs of lysosomal repair and lysosomal biogenesis in macrophages can have atheroprotective effects. A class of important regulators of the autophagy-lysosomal system are cytosolic lectins. When the lysosome membrane becomes damaged, Galectin-3 (Gal3) recognizes the damage and recruits autophagic receptors and delivers the cargo to autophagosomes. Additionally, Gal3 may recruit the ESCRT complex and facilitate the repair and restoration of lysosomes. We hypothesized that Gal3 provides a protective role in atherogenic macrophages, by repairing or removing damaged lysosomes, thus preventing the relevant deleterious signaling pathways such as inflammasome activation and apoptosis. We utilized Gal3-KO macrophages and mice to study the role of Gal3 in atherosclerosis. First, we show that Gal3-KO macrophages are highly sensitive to lysosome membrane damaging agents including atherogenic oxLDL and cholesterol crystals, exhibiting decreased autophagy and protein aggregate clearance compared to the WT controls. Furthermore, the recruitment of lysosome repair mechanisms involving the ESCRT complex are blunted. Such blunting of critical repair mechanisms results in increased macrophage apoptosis and inflammasome/IL-1β activation. These findings are also recapitulated in vivo, where bone marrow transplants of atherogenic LDLR-null mice with Gal3-deficient bone marrow results in increased plaque size and altered plaque composition with significant macrophage accumulation, apoptosis, and necrotic core formation, characteristic features of advanced plaques. Overall, our data demonstrate important roles for Gal3 in macrophage lysosome repair mechanisms which are a critical response against atherosclerosis.
Abstract 528: Characterization Of Sitosterolemia-causing Mutations In The Extracellular Domain Of Abcg8
Authors: Chauhan Isha , Williams Kori , Poole Brittney , Zelows Mikala , Graf Gregory A
Published: November 2022

Background:
ABCG5 and ABCG8 form a heterodimer that promotes biliary secretion and opposes intestinal absorption of cholesterol and phytosterols. Whole-genome and exome sequencing has revealed over 2000 variants of ABCG5 and ABCG8, many of which are predicted to be pathogenic or likely pathogenic. Missense mutations in clinically confirmed cases of Sitosterolemia are of particular interest for their potential to reveal structure-function relationships in ABCG5 ABCG8 and other ABC transporter family members. Mutations in ABCG5 ABCG8 with clinically confirmed Sitosterolemia generally cluster within the ATP binding cassette and transmembrane spanning segments that form the substrate-binding domain. However, four disease-causing mutations occur in the short extracellular loop in ABCG8, suggesting that these mutations may reveal novel elements of the ABCG5 ABCG8 function.

Methods:
Mutants of ABCG8 were generated by site-directed mutagenesis. Plasmids encoding normal ABCG5 and normal or mutant ABCG8s were co-transfected into human Huh7 hepatocytes, lysates prepared, and analyzed by SDS-PAGE and immunoblot analysis. Maturation and trafficking of the ABCG5 ABCG8 heterodimer beyond the endoplasmic reticulum was assessed by the appearance of the higher molecular weight, mature form of each glycoprotein.

Results:
Mutants G574R, G575D, and L596R failed to support trafficking of ABCG5 ABCG8 and were designated as Class II mutants causative in Sitosterolemia. Only G574E supported maturation of the ABCG5 ABCG8 transporter in cultured human hepatocytes and remains unclassified.

Conclusion:
Formation and trafficking of the ABCG5 ABCG8 heterodimer is dependent on key residues within the third extracellular loop in ABCG8. Maturation of ABCG5 ABCG8 is tolerant of substitutions in glycine 574, including the addition of a larger and negatively charged residue. However, neither substitution forms a functional ABCG5 ABCG8 transporter.
Abstract 557: Stretching With Ankle Dorsiflexion Splint Improves Measures Of Microvascular Reactivity And Oxygen Extraction In Patients With Peripheral Artery Disease
Authors: Martenson Matthew , Delp Judy M , Caldwell Jacob , Kaelin Lawrence , Hakaim Albert , Panton Lynn
Published: November 2022

Peripheral Artery Disease (PAD) is a vascular disease that affects over 8 million Americans. PAD patients commonly have difficulty walking due to pain in the legs during physical exertion. Supervised walking therapy has been recommended to improve walking performance and attenuate disease progression; however, this therapy remains underutilized in PAD patients. Recently, there has been evidence to suggest that stretching accomplished via an ankle dorsiflexion splint may improve vascular health and walking performance in PAD; however, it is currently unknown how this protocol affects tissue oxygenation (StO
2
). The purpose of this study was to determine the impact of 4 wks of ankle dorsiflexion stretching on measures of tissue oxygenation in patients with PAD. Thirteen PAD patients (67.7 ± 7.9 years) were randomized to a stretch (n = 9) or a non-stretch control (n = 4) group. Patients in the stretch were instructed to wear the splint for 30 min/day, 5 days/wk for 4 wks. A 5-min vascular occlusion test was administered before and after the intervention. Measures of StO
2
recovery were calculated following the vascular occlusion test and while completing a 6-minute walk test (6MWT). All measures of StO
2
were derived via Near-Infrared Spectroscopy. Repeated measures ANOVA was used to analyze data. There was a significant improvement in StO
2
recovery in the stretch group compared to the control (Stretch: 0.35 ± 0.25 %/sec vs 0.53 ± 0.29 %/sec; Control: 0.57 ± 0.43 %/sec vs 0.32 ± 0.19 %/sec; p = 0.002). During the 6MWT, patients completing the stretching protocol significantly improved oxygen extraction in the first minute of the test (-0.63 ± 0.05 StO
2
vs -0.73 ± 0.09 StO
2
); however, this improvement was not significantly different compared to changes in the control group. These results indicate an improvement in microvascular reactivity and oxygen extraction following 4 wks of splint-based stretching. This stretching intervention holds potential therapeutic value for PAD patients and warrants further investigation.
‘Belief’ and Anthropology, in Use and in Theory
Authors: Streeter Joseph
Published: November 2022

This essay examines the standing of three important and widely accepted criticisms of the use of the concept of belief within the anthropology of religion. It does so by examining whether these criticisms track the historical use of the concept within the discipline, that is, whether the problematic implications that they associate with the concept can reasonably be inferred from the historical use of the words ‘believe’ and ‘belief’ by anthropologists. It argues that the criticisms do not meet this standard, and that we therefore have reason to think that they are pseudo-problems, which have no legitimate claim on our attention. It concludes by suggesting some reasons why these apparently arbitrary concerns about the concept of belief should have arisen, looking first at some discussions of the meaning of the terms ‘believe’ and ‘belief,’ and second at the reception of Rodney Needham’s 1972 monograph Belief, Language, and Experience.
Searching for Borders of Freedom: Foreign Trips of Czechoslovak Musicians and Athletes between 1948–1989
Authors: Mücke Pavel , Krátká Lenka
Published: November 2022

In the era of the “people’s democratic” and later “socialist” Czechoslovakia (1948–1989), foreign trips, and even more business trips because of their financial impact on one’s life, represented privileges that were to be granted by the ruling elites only to reliable and closely-watched Czechoslovak citizens in the name of the “public interest.” At the same time, the business trips became a kind of litmus test of the loyalty and the “importance” of the travelling (in case of disapproval, non-travelling) individual in the regime’s eyes. In this context, the paper focuses on the lived experience of musicians and athletes concerning foreign trips. Finally, it discusses the potential contradictions between some popular stories in the Czech collective memory on the one hand and more detailed micro-historical probes on the other hand, which demonstrate the interpretive richness of the discussed issues.
Supporting Curriculum Innovation in Integrated STEM for Secondary Teachers in Australia
Authors: Anderson Judy , Tytler Russell , Williams Gaye
Published: November 2022

Given concerns in the Australian context about students’ attitudes and declining participation in STEM subjects in the senior secondary years, a range of initiatives have been developed to support teacher design of innovative STEM curriculum and pedagogy. The STEM Academy program worked with interdisciplinary teams of secondary STEM teachers to develop teacher capacity to create real-world, challenging problems to engage and motivate their students. Questionnaire and interview data collected from one cohort of 70 teachers from twelve schools, three program facilitators, and school leaders provided a case study revealing themes about the nature of the journey, the role of the program and the processes and varied nature of schools’ STEM curriculum innovations. An innovation framework was used to make sense of teachers’ journeys towards effective and sustainable STEM practices leading to the identification of three models that reflected different approaches to the challenge of representing STEM within subject-based curricular settings.
The analgesic Efficacy and Safety of Topically Applied Tramadol in Peritonsillar Space During Pediatric Adenotonsillectomy: A Meta-Analysis
Authors: Kang Yun Jin , Cho Jin-Hee , Hwang Se Hwan
Published: November 2022

Objectives:

To evaluate the efficacy of topically applied tramadol in peritonsillar area for pain control and induction of perioperative morbidity in children undergoing adenotonsillectomy.

Methods:

Two authors independently searched the databases (MEDLINE, SCOPUS, and Cochrane databases) up to April 2022 for randomized controlled trials comparing the efficacy of an intraoperative peritonsillar administered tramadol with placebo or other agents only in pediatric patients. The outcomes were postoperative pain scores, time to take the first pain reducing drugs, and postoperative nausea/vomiting.

Results:

Our analysis was based on 17 trials. Peritonsillar administered tramadol significantly reduced postoperative pain at less than 20 minutes (SMD −1.5852, 95% CI [−2.3900; −0.7804]), 1 hour (SMD −1.5811, 95% CI [−2.3400; −0.8222]), 8 hours (SMD −1.0258, 95% CI [−1.8792; −0.1724]), 16 hours (SMD −0.8397, 95% CI [−1.4266; −0.2529]), and 1 day (SMD −1.0110, 95% CI [−1.5213; −0.5007]) and the time to take the first analgesic drug (SMD 1.6565, 95% CI [0.2838; 3.0293]) compared with the placebo. However, tramadol showed no significant difference on postoperative pain relief effects and the time to take the first analgesic drug compared to the other topical agents (bupivacaine, lidocaine, ketamine, and dexamethasone). Tramadol did not cause significant postoperative nausea and vomiting.

Conclusions:

This study demonstrated that intraoperatively applied topical tramadol could control postoperative pain and reduce the analgesic uptake compared with a control. However, the efficacy of tramadol in pain control was similar to other agents in pediatric adenotonsillectomy.
The Evaluation and Management of Pediatric Diabetic Ketoacidosis: A Comprehensive Review
Authors: Bonadio William
Published: November 2022

Diabetic ketoacidosis (DKA) is a common, serious acute complication in children with diabetes mellitus (DM). DKA can accompany new-onset type 1 insulin-dependent DM, or it can occur with established type 1 DM, during the increased demands of an acute illness or with decreased insulin delivery due to omitted doses or insulin pump failure. In addition, DKA episodes in children with type 2 DM are being reported with greater frequency. Although the diagnosis is usually straightforward in a known diabetes patient with expected findings, a sizable proportion of patients with new-onset DM present with DKA. The purpose of this comprehensive review is to acquaint clinicians with details regarding the pathophysiology, treatment caveats, and potential complications of DKA.