bluuebunny/crossref_metadata_2025_split huggingface dataset:
Abstract 55: Romiplostim Treatment Restores GPVI Expression and Platelet Counts in Mice Lacking the ITIM-Containing Receptor G6b-B
Authors: Senis Yotis A , Mazharian Alexandra , Heising Silke , Watson Steve P 
Published: July 2021

We recently demonstrated that the immunoreceptor tyrosine-based inhibition motif (ITIM)-containing receptor G6b-B plays a critical role in regulating platelet homeostasis. Mice lacking G6b-B exhibit a complex phenotype that includes severe macrothrombocytopenia, platelet surface immunoglobulins, reduced expression of the collagen activation receptor GPVI, and aberrant platelet function. In this study, we tested the effects of the thrombopoietin-mimetic romiplostim in ameliorating defects seen in G6b-B-deficient mice. Subcutaneous administration of romiplostim (100 μg/kg romiplostim every three days) restored platelet counts to normal levels in G6b-B-deficient mice within two weeks. Platelet surface GPVI expression was also elevated by five-fold in romiplostim-treated G6b-B-deficient mice compared with platelets from mice treated with vehicle alone, restoring platelet reactivity to the GPVI-specific agonist collagen-related peptide (CRP). In contrast, the same romiplostim regimen induced a 40% reduction in platelet surface GPVI expression in control mice, resulting in comparable levels of GPVI in romiplostim-treated G6b-B-deficient and control mice. Megakaryocyte counts were dramatically increased in the bone marrow and spleen of romiplostim-treated G6b-B-deficient and control mice, accompanied by severe myelofibrosis in both genetic backgrounds. Bone marrow-derived megakaryocytes from G6b-B-deficient mice grew normally in vitro in the presence of thrombopoietin, but exhibited reduced proplatelet formation on a fibrinogen-coated surface. Findings from this study demonstrate that romiplostim can be used to restore GPVI expression in addition to platelet counts in G6b-B-deficient mice, but has severe side-effects on bone marrow and spleen myelofibrosis. This approach can now be applied to further investigate the functional role of G6b-B in regulating platelet counts and reactivity.
This work was funded by the British Heart Foundation.
Abstract 617: Intestine-Specific MTP Deficiency with ACAT2 Gene Ablation Lowers Acute Cholesterol Absorption With Chylomicrons and High-Density Lipoproteins
Authors: Iqbal Jahangir , Boutjdir Mohamed , Rudel Lawrence L , Hussain M Mahmood 
Published: July 2021

Intestinal cholesterol absorption involves chylomicron and high density lipoprotein (HDL) pathways. Microsomal triglyceride transfer protein (MTP) and ATP binding cassette family A protein 1 (ABCA1) are critical for cholesterol transport by these pathways, respectively. Additionally, acyl Co-A:cholesterol acyltransferase 2 (ACAT2) plays an important role in cholesterol absorption. Intestinal MTP ablation significantly increased intestinal triglyceride and cholesterol levels and reduced their acute absorption. In contrast, ACAT2 deficiency had no effect on triglyceride absorption but significantly reduced cholesterol absorption. Individual deficiencies of ACAT2 and MTP reduced cholesterol absorption with chylomicrons. We hypothesized that their combined deficiency would increase cholesterol secretion with HDL; unexpectedly, their deficiency reduced secretion with both chylomicrons and HDL. Further, we observed significant reductions in intestinal ABCA1 expression in combined deficient mice. Thus, free cholesterol is unavailable for secretion by the HDL pathway in these mice. We speculate that reductions in ABCA1 expression and HDL secretion might be secondary to massive triglyceride accumulation associated with intestinal MTP deficiency.
Besides its role in cholesterol absorption, ACAT2 deficiency causes mild hypertriglyceridemia and reduces steatosis in mice fed high cholesterol diets by increasing hepatic lipoprotein production by unknown mechanisms. We show that this phenotype is preserved in the absence of intestinal MTP in ACAT2 deficient mice fed a Western diet. Further, we observed increases in hepatic MTP activity in these mice. Thus, ACAT2 deficiency might increase MTP expression to avoid steatosis. Therefore, ACAT2 inhibition might avert steatosis associated with high cholesterol diets by increasing MTP expression.
Abstract 62: Cholesterol Efflux Pathways in Dendritic Cells Suppress Autoimmune Responses
Authors: Westerterp Marit , Gautier Emmanuel L , Ganda Anjali , Randolph Gwendalyn J , D'Agati Vivette D , Yvan-Charvet Laurent , Tall Alan R 
Published: July 2021

Patients with auto-immune disorders have low HDL levels. Mice deficient in genes regulating cholesterol homeostasis, such as liver X receptor (LXR) or apolipoprotein A1 (apoA1), show an autoimmune phenotype. LXR regulates the expression of ATP Binding Cassette A1 and G1 (ABCA1 and ABCG1) that mediate cholesterol efflux to apoA1 and HDL. ABCG1 is highly expressed in dendritic cells (DCs). We hypothesized that ABCA1 and ABCG1 regulate autoimmunity.
On a chow diet, 40 weeks old Abca1-/-Abcg1-/- mice showed enlarged lymph nodes (LNs), increased plasma auto-antibodies to dsDNA, and glomerulonephritis, with characteristics typical for lupus nephritis. Using the Cre loxP system, we investigated whether these effects were due to Abca1/g1 deficiency in T-cells, macrophages, or DCs. Only Abca1/g1 deficiency in DCs in CD11cCreAbca1fl/flAbcg1fl/fl mice replicated the auto-immune phenotype found in Abca1-/-Abcg1-/- mice. This suggests a major role for DC cholesterol homeostasis in autoimmunity.
DCs present antigens to T-cells, leading to their activation. CD11cCreAbca1fl/flAbcg1fl/fl mice showed increased T-cell activation in blood, spleen, and LNs. After immunization, DCs from CD11cCreAbca1fl/flAbcg1fl/fl mice showed increased antigen presentation to T-cells in vitro and in vivo. CD11cCreAbca1fl/flAbcg1fl/fl mice had increased CD80+ DCs. CD80 is a co-stimulatory molecule required for antigen presentation. Abca1/g1 deficiency in DCs increased endosomal cholesterol accumulation in vitro. Ligands for Toll like receptor (TLR) 3 and 4 increased CD80 mRNA in CD11cCreAbca1fl/flAbcg1fl/fl compared to Abca1fl/flAbcg1fl/fl DCs, where the effect of ligands for TLR3>TLR4. Cholesterol depletion by cyclodextrin decreased CD80 mRNA and antigen presentation in CD11cCreAbca1fl/flAbcg1fl/fl DCs in vitro. The increased CD80 mRNA in CD11cCreAbca1fl/flAbcg1fl/fl DCs was also reversed by a type I interferon (IFN) antibody, suggesting excessive signaling by the known TLR3-IFN-CD80 axis. These studies show for the first time a role for cholesterol efflux pathways in DCs in maintaining immune tolerance.
Abstract 622: Development of an Antioxidized Phospholipid Neutralizing Antibody as an Atheroprotective Therapeutic Agent
Authors: Cherepanova Olga A , Owens Gary K 
Published: July 2021

Atherosclerosis is a chronic disease of the arterial wall, which contributes to >40% of all deaths in developed societies. Widespread use of statins and life style modifications within developed nations have resulted in modest reductions in the incidences and severity of atherosclerotic disease, but as yet there is no clear evidence that statins can prevent late stage clinical consequences of atherosclerosis including plaque rupture with possible myocardial infarction or stroke.
Atherosclerosis is a complex disease characterized by two key processes, lipid deposition and inflammation within the arterial wall, and oxidative stress providing the link between the two. Oxidized phospholipids (OxPL), such as oxidized PAPC (1-palmitoil-2-arachodonoyl-sn-glycero-3-phosphorylcholine) and its derivatives are the biologically active components of minimally oxidized LDL, whose role in cardiovascular diseases is well recognized. Atherosclerotic ApoE knockout (KO) mice fed a high fat Western diet develop antibodies (Abs) to OxPL, and hybridoma B cell lines producing natural auto-Abs against OxPL have been successfully generated and characterized. However, as yet, no studies have been reported demonstrating that treatment with OxPL neutralizing Abs can be used to prevent or reverse atherosclerosis.
Of major importance, we recently generated an anti-OxPL auto-Ab (10C12) that demonstrates potent OxPL neutralizing activity in vitro. Sequence analysis showed that 10C12 Ab is structurally different from previously described anti-OxPL EO6 type neutralizing Abs. Moreover, we found that 10C12 Ab injections (IP, twice a week) in ApoE KO mice fed a Western diet for four weeks resulted in: 1) high circulating titers of Abs to OxPL as measured by ELISA; 2) significantly decreased accumulation of macrophages within the brachiocephalic artery, a site of advanced plaque formation in mice; and 3) a decreased OxPL-induced inflammatory response following IP injection of OxPL as determined by analysis of inflammatory gene expression in abdominal lavage cells.
Results of our work give insight into the role of OxPL in atherosclerosis and lead to potential novel therapeutic approaches to treat it or prevent clinical complications of atherosclerosis.
Abstract 651: Phenotype Shifts in Vascular Smooth Muscle Cells Induced by a Low-Carbohydrate High-Protein Diet
Authors: Koury Olivia H , Vuckovic Dajana , Bergdahl Andreas 
Published: July 2021

The growing rate of obesity is of concern as the condition is associated with increased risk for cardiovascular disease (CVD). Diets that reduce or eliminate carbohydrates, while increasing fat and/or protein intake (Atkin’s Diet) are growing in popularity for rapid weight loss. Despite their effectiveness, recent studies have implicated these low-carbohydrate high-protein (LCHP) diets with an increased incidence of CVD and greater fatty deposits in the aortic wall than a traditional western diet (WD).
We assessed the hypothesis that LCHP diets result in greater vascular smooth muscle cell (VSMC) dedifferentiation and a more pro-atherosclerotic environment when compared to a control diet (CON) or WD.
Experiments were performed on aorta and plasma obtained from male Apolipoprotein E-knockout mice that were fed either regular chow (CON), WD or LCHP diet for 6 weeks. Immunoblotting was used to determine VSMC dedifferentiation as a marker of disease stage in isolated vessels using proteins calponin, alpha-actin, and TRPC1. Plasma lipid concentrations were obtained by liquid chromatography - mass spectrometry (LC-MS).
Calponin was significantly downregulated in the LCHP diet versus both the WD and CON, with no difference detected between WD and CON. There was a trend towards a decreased level of alpha-actin and a higher TRPC1 level in the LCHP diet compared with CON (no differences between CON and WD). LC-MS data revealed significantly higher plasma levels of certain triglycerides in LCHP than WD.
This data indicates that the LCHP diet results in a more synthetic phenotype of SMC as indicated by the diminished contractile protein expression (calponin and alpha-actin) and increased TRPC1, when compared to CON and WD. Additionally, the LCHP diet results in an abnormal lipid profile when compared to the WD; a traditional high-risk diet. Taken together, this data suggests that the LCHP diet results in an increased pro-atherosclerotic environment, even when equated to the WD in terms of cholesterol intake. This novel information may require attention from both clinicians and dieters when evaluating diet choices.
Abstract 667: Ergosterol or Brassicasterol Accumulations in ABCG8 Knockout Mice Do Not Account for Xenosterol Toxicity
Authors: Patel Shailendra B 
Published: July 2021

Xenosterol accumulation mice deficient in sterolin function leads to significant toxicity, with infertility, decreased body fat accumulation, macrothrombocytopenia, cardiac fibrosis and premature death1,2. Although sitosterol has been shown to have some biological effect in tissue culture, only stigmasterol has been shown to have a potent biological effect, by activating the transcriptional factor, Lxr3. In patients with sitosterolemia, feeding shell-fish sterols led to accumulation of shell-fish sterols in their plasma4, suggesting any xenosterol could accumulate, if fed, to mammals deficient in Abcg5 or Abcg8.
Fungi do not utilize cholesterol, but instead use ergosterol as the primary membrane sterol. To delineate whether ergosterol could accumulate in Abcg8 knockout mice, and lead to toxicity, we designed a diet that was supplemented with highly enriched ergosterol (>98% purity) and fed this to Abcg8 knockout mice.
Over a 12-week period, both male and female Abcg8 knockout mice fed an ergosterol-enriched diet gained normal amounts of weight, body fat, showed no disturbances in tail-cuff measured blood pressure, and showed no abnormalities of platelet counts or volumes, blood glucose, or plasma cholesterol.
Fertility testing showed no abnormalities.
Dramatically, analyses of plasma from these mice showed no accumulation of ergosterol, but a dramatic increase in plasma brassicasterol, with levels reaching 80mg/dL in plasma. Gene expression analyses of livers did not show any consistent patterns, although Lxr target genes were not up-regulated. These data do not support the concept that brassicasterol accumulation, at levels of 80mg/dL in plasma, account for the xenosterol-mediated toxicity observed. Additionally, the intestine shows powerful 1st pass detoxification of ergosterol by converting it to brassicaterol by using (presumably) dehydrocholesterol Δ7 reductase enzyme and highlights the importance of intermediary metabolism enzymes as part of the defense against xenosterols.
1. McDaniel, A.L.et al Am J Pathol, 2013. 182(4): p. 1131-8.
2. Solca, C. et al J Lipid Res, 2013. 54(2): p. 397-409.
3. Yang, C.et al J. Clin. Invest., 2004. 114(6): p. 813-22.
4. Gregg, R.E.et al J. Clin. Invest., 1986. 77(6): p. 1864-72.
Abstract 75: Characterization of Knock-in Mice Harboring a Variant of Endothelial Protein C Receptor with Impaired Ability to Bind Protein C: Impact on Coagulation and Hematopoiesis
Authors: Pepler Laura , Dwivedi Dhruva J , Liaw Patricia C 
Published: July 2021

The endothelial protein C receptor (EPCR) binds to protein C (PC) and increases the rate of activated protein C (APC) generation by the thrombin-thrombomodulin complex at the endothelial cell surface. APC exerts anticoagulant, anti-inflammatory, and cytoprotective effects, many of which are EPCR-dependent. The physiologic importance of EPCR is also demonstrated in EPCR knockout mice, which show placental thrombosis and early embryonic lethality. EPCR is also highly expressed on hematopoietic stem cells (HSC), however there is no known biological role for EPCR in this cell type.
Currently, there are no animal models to study the biological role of EPCR independent of its interaction with PC/APC. In this study, we generated a knock-in mouse model harboring a variant of EPCR (R84A) which lacks ability to bind to PC/APC. We hypothesize that loss of PC/APC binding to EPCR will result in a procoagulant and pro-inflammatory phenotype.

EPCR
R84A/R84A
mice are viable, have a normal lifespan and show no evidence of spontaneous thrombosis. Histological analysis of EPCR
R84A/R84A
mice identified a splenic disorder, characterized by splenomegaly and extramedullary hematopoiesis. Flow cytometric analysis of the spleen from EPCR
R84A/R84A
mice revealed a significant increase in the percentage of CD34+ cells, representing HSCs (2.4 ± 0.1% for EPCR
R84A/R84A
mice and 1.4 ± 0.2% for WT mice; P<0.05). To initiate thrombin generation in mice, an intravenous injection of FXa was administered. Plasma APC levels of FXa challenged EPCR
R84A/R84A
mice were reduced by 150% and thrombin-antithrombin levels (TAT) (an indicator of thrombin generation) were increased by 100% when compared to WT mice. The elevated TAT levels in FXa challenged EPCR
R84A/R84A
mice were accompanied by an increase in the size and number of fibrin clots in the lungs.

EPCR
R84A/R84A
mice are viable suggesting that defects in EPCR that impair PC binding do not affect embryogenesis or development. Introduction of the R84A mutation in EPCR results in impaired PC activation, and a procoagulant phenotype upon thrombotic challenge. Enlargement of the spleen in EPCR
R84A/R84A
mice suggests that EPCR may play a biological role in the regulation of hematopoiesis.
Abstract 121: Pro-inflammatory Cytokine Ifng Modulates Hepatic Sortilin Expression and Lipid Metabolism.
Authors: Pirault John , Polyzos Konstantinos , Ketelhuth Daniel F , Hansson Göran K 
Published: July 2021

Rationale:
Hypercholesterolemia and immunity are two major risk factors for cardiovascular diseases (CVDs). Yet, we reported increased atherosclerosis upon depletion of regulatory T lymphocytes (Tregs). The effect was associated with increased hepatic inflammation and reduction of Sortilin expression and lipid uptake in the liver.

Objective:
To define how inflammatory milieu in the liver can modulate Sortilin and lipid metabolism.

Methods:
To reproduce the inflammatory milieu, hepatocytes (AML-12) were treated in vitro with IFNg. Expression of genes and proteins of interest were followed by qPCR and western blot. In silico method was used to find binding sites of signal transducer and activator of transcription (STAT1) on Sortilin, confirmed later by chromatin immune precipitation assays (Chip). Lipid uptake by hepatocytes was assessed via incubation of cells with radioactive lipoproteins.

Results:
Culture of AML-12 cells with IFNg induced the phosphorylation of STAT1 showing an active signaling pathway. In the same inflammatory conditions, Sort1 mRNA is decreased meanwhile its inhibitor (Atf3) expression is increased. Kinetic experiments revealed the reduction of Sortilin after 12 hours of culture, suggesting a post-transcriptional regulation of Sort1 by STAT1.

In silico analysis revealed putative binding sites for STAT1 on Sortilin gene which was confirmed by chromatin immunoprecipitation assay (Chip).
IFNg treated hepatocytes that were incubated with radioactive lipoproteins demonstrated a reduced uptake capacity of VLDL and LDL particles compared to control cultures.

Conclusion:
All together, these results suggest that inflammation through production of IFNg is able to directly modulate the lipid metabolism in hepatocytes by acting on Sortilin expression.
Abstract 140: Nuclear Targeting Apelin Induces Phenotypic Transition of Vascular Smooth Muscle Cells
Authors: Bochaton-Piallat Marie-Luce , Chaabane Chiraz , Brun Cécile , König Stéphane , Audigier Yves , Baertschi Alex J 
Published: July 2021

Apelin, and its receptor APJ, are a peptidic system playing a crucial role in vascular diseases. However, the role of apelin in atherogenesis and smooth muscle cell (SMC) proliferation remains unclear. We isolated 2 distinct SMC phenotypes from porcine coronary artery: spindle-shaped (S) and rhomboid (R). Biological features of R-SMCs (i.e. enhanced proliferative and migratory activities as well as poor level of differentiation) explain their capacity to accumulate into the intima. We identified S100A4 as being a marker of the R-SMCs in vitro and of intimal SMCs, both in pig and human.
The aim of our study was to investigate the effects of apelin on SMC phenotypic transition and S100A4 expression.
Apelin was highly expressed in R-SMCs including in their nucleus. P-SORT software analysis of preproapelin sequence suggested that N-terminal truncated apelin may target the nucleus, and we confirmed this in SMCs by overexpression of mutated preproapelin-His-tag. Transfection of mutated preproapelin-His-tag encoding plasmid in differentiated S-SMCs induced a transition towards a R-phenotype associated with increased proliferative activity, downregulation of SMC differentiation markers, and increased nuclear expression and release of S100A4. In contrast, transfection of S-SMCs with wild type preproapelin-His-tag encoding plasmid did not induce nuclear targeting of apelin or S100A4, and did not change the S-phenotype. Stimulation of S-SMCs with PDGF-BB, known to induce a transition to the R-phenotype, yielded nuclear targeting of both apelin and S100A4. In vivo, Apelin was expressed in SMC nuclei of stent-induced intimal thickening while its expression in the media was mainly cytoplasmic.
Our results suggest that nuclear targeting of apelin in SMCs acts on S100A4 expression and release, cell proliferation and differentiation. The pathophysiological consequences of this retargeting could be instrumental in the understanding of artherosclerosis.
Abstract 169: Impaired Shear Stress-induced Endothelial Nitric Oxide Production in High Glucose Condition is Restored by Inhibiton via NADPH Consumption by Polyol Pathway Activation
Authors: Umemoto Tomio , Kuroki Masatoshi , Ueba Hiroto , Kawakami Masanobu , Fujita Hideo , Momomura Shin-ichi 
Published: July 2021

Endothelial dysfunction leading to cardiovascular disease risk involves a decrease in nitric oxide (NO) production. In physiological conditions shear stress is a potent stimulation of endothelium-derived NO production and flow mediated NO production is regulated by the activation of endothelial NO synthase (eNOS). In endothelial cells, eNOS, aldose reductase (AR), a rate limiting enzyme of polyol pathway, and glutathione reductase (GR) share a NADPH as an obligate cofactor. In diabetec condition intracellular polyol pathway is activated and this may decrease shear stress-induced endothelial NO production and increase intracellular oxidative stress via inhibition of eNOS and GR by NADPH consumption. Therefore we investigated whethter AR inhibitor epalrestat improved endothelial NO production under high glucose condition to elucidate the mechanism of endothelial dysfunction in diabetes.
We incubated human umbilical vein endothelial cells (HUVECs) in normal (5mM) and high (30mM) glucose condition for 72 hours, with or without epralrestat, or 100U/ml superoxide dismutase (SOD), respectively. After exchange of medium for Krebs’ buffer, HUVECs were exposed to 12dyne/cm2 steady laminar fluid shear stress for 5 minutes. NO release from HUVECs was measured as NO2 using a NOx analyzing HPLC system by Griess reaction. Next we harvested the cells in lysis buffer and analyzed phosphorylation of Akt (shear induced intracellular signal transduction) and eNOS by western blotting, and measured intracellular 8-OHdG and ratio of NADPH/NADP.
In high glucose condition NO2 was decreased and 8-OHdG increased compared to low glucose. NO2 was restored and 8-OHdG was reduced by epalrestat significantly (p<0.01, p<0.05, respectively, vs. high glucose condition). In SOD-treated HUVECs, NO2 was not restored (n.s. vs. high glucose condition) despite of complete reduction of 8-OHdG (p<0.01). Both Akt and eNOS phosphorylation by shear stress was affected neither by high glucose, epalrestat nor SOD. Intracellular NADPH/NADP ratio was decreased in high glucose condition, but this reduction was restored by epalrestat.
These results showed that polyol pathway activation plays a key role in endothelial NO production under high glucose condition via a cofactor NADPH.