bluuebunny/crossref_metadata_2025 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 555: Bone Morphogenetic Protein Modulator BMPER Induces Angiogenesis via Inhibition of Thrombospondin-1 and Activation of the Fibroblast Growth Factor Signaling Pathway
Authors: Esser Jennifer S , Rahner Susanne , Deckler Meike , Bode Christoph , Moser Martin
Published: July 2021

Introduction:
Bone morphogenetic proteins (BMP) play a key role in vascular development. Previously, we have identified BMP endothelial cell precursor-derived regulator (BMPER), an extracellular BMP modulator, to increase the angiogenic activity of endothelial cells in a concentration-dependent manner. In this project we now investigate how the BMPER effect is mediated by key molecules of angiogenesis.

Methods and Results:
To assess the effect of BMPER on angiogenesis-related molecules we performed an angiogenesis antibody array with BMPER-stimulated human umbilical venous endothelial cells (HUVECs) and vice versa with BMPER-silenced HUVECs compared to control conditions, respectively. We detected increased protein expression of the anti-angiogenic thrombospondin-1 (TSP-1) 48 hours after siBMPER transfection and, consistently, decreased TSP-1 expression after stimulation with BMPER (60 ng/ml; 39% ± 7.3 N=4). Furthermore, the pro-angiogenic protein bFGF was increased after BMPER stimulation, which was confirmed by realtime-PCR and western blot analysis (288.8% ± 74.8 N=3). Additionally, we detected increased FGF receptor-1 protein expression (137.7% ± 0.4 N=3) as well as FGF signaling pathway activation. Next, we investigated the interaction of BMPER and the FGF signaling pathway in endothelial cell function. BMPER stimulation increased HUVEC angiogenic activity in matrigel, migration and spheroid assays and concomitant inhibition of FGF signaling by an anti-bFGF antibody effectively inhibited the pro-angiogenic BMPER effect. Silencing of BMPER decreased the expression of FGFR1 and, accordingly, stimulation of BMPER-silenced cells with bFGF showed decreased angiogenic endothelial cell activity (65%) compared to control. The angiogenic activity of bFGF was also reduced in C57BL/6_Bmper +/- mice as assessed in the matrigel plug assay. Ex vivo aortic ring assays of C57BL/6_Bmper +/- mice confirmed a specific effect for bFGF but not for VEGF.

Conclusion:
In summary, BMPER inhibits the expression of the anti-angiogenic TSP-1 and increased the expression as well as activation of the pro-angiogenic FGF signaling pathway, which overall lead to the promotion of angiogenesis.
Abstract 560: Surface Glycoengineering of Mesenchymal Stem Cells and Cardiosphere-Derived Cells Enhances Selectin-Mediated Cell Rolling and Tethering
Authors: Lo Chi Y , Canty John M , Neelamegham Sriram
Published: July 2021

Background:
Of the current stem cell delivery routes, vascular injection has the advantage of not being limited by the poor vascular accessibility of the donor cells as in the cases of intramyocardial injection and cell-sheet placement. However, there is concern about the lower cell retention in the heart; thus, we access the hypothesis that modifying the cell surface glycosylation on MSCs and CDCs can enhance their interaction to inflammatory sites by increasing interactions with endothelial selectins.

Methods:
Two approaches were taken to address the hypothesis. First, the non-covalent coupling of a P-selectin glycoprotein ligand-1 (PSGL-1) fusion protein called 19Fc[FUT7
+
] to MSCs and CDCs is performed using two types of protein G lipid conjugates, either PPG or DPG. Second, we investigated the increased of α(1,3) fucose on the cell surface glycans by overexpression of three fucosyltransferases (FUT6, FUT7, and FUT9).

Results:
MSCs functionalized with 19Fc[FUT7
+
] were captured onto stimulated endothelial cell monolayers at wall shear stresses up to 4 dyn/cm
2
. Both MSCs and CDCs could be captured onto P-selectin substrates up to 6 dyn/cm
2
, (Table 1). In the case of fucosyltransferase overexpression, FUT7 improved the number of both MSCs and CDCs captured onto P- and E-selectin substrates. FUT9 also significantly improved the number of CDCs captured to E-selectin.

Conclusion:
The current work presents a robust strategy for enabling leukocyte-like capture and rolling under constant flow conditions to enhance cell delivery efficiency thereby increasing tissue engraftment. This method could be employed to increase tissue engraftment of cells used in stem cell therapy.
Abstract 59: Fibronectin Extradomain A Modulates Atherosclerotic Lesion Progression in Hypercholesterolemic Mice Through Toll-Like Receptor 4
Authors: Gandhi Chintan , Prakash Prem , Ahmad Ajmal , Chauhan Anil
Published: July 2021

Background and Hypothesis:
Fibronectin (FN) containing the alternatively-spliced extra domain A (EDA
+
-FN) is absent in the arteries or circulation of healthy humans and mice, but specifically expressed in the endothelium of atherosclerotic arteries and elevated in circulation during pathological settings including stroke and atherosclerosis. We tested the hypothesis that EDA
+
-FN exacerbates atherosclerosis.

Model and Methods:
We generated EDA
hi/hi
/ApoE
-/-
mice (over express EDA
+
-FN in plasma) and EDA
-/-
/ApoE
-/-
mice (EDA-deleted allele of FN). ApoE
-/-
mice, which express EDA
+
-FN in plasma (~3-5 μg/mL) similar to levels found in human pathological settings including stroke and atherosclerosis, were used as control. Male mice (n=14/group) were fed a high-fat Western diet beginning at 6 weeks until they were sacrificed at 5 and 8 months of age. Extent of atherosclerosis was evaluated in whole aortae (Oil Red O stain) and in the cross section area of the aortic sinus (Verhoeff-Van Gieson stain). Markers of inflammation were quantified by immunohistochemistry.

Results:
We report that atherosclerotic plaque formation in the aortae and aortic sinus of EDA
hi/hi
/ApoE
-/-
mice were increased by 2-fold, concomitant with increased plaque inflammatory content (neutrophils, macrophages, and NF-κB p65 expression; P<0.01 vs. ApoE
-/-
). Conversely, EDA
-/-
/ApoE
-/-
mice had smaller plaques (P<0.01 vs. ApoE
-/-
mice), concordant with decreased inflammatory content within plaque. Because EDA
+
-FN activates toll-like receptor 4 (TLR4)
in vitro
, we investigated the role of TLR4 in the EDA
+
-FN-mediated accelerated atherosclerosis. Genetic ablation of TLR4 in EDA
hi/hi
/ApoE
-/-
mice partially reversed exacerbated atherosclerosis (P<0.05 vs. EDA
hi/hi
/ApoE
-/-
mice), whereas TLR4 deficiency in EDA
-/-
/ApoE
-/-
had no effect. Total cholesterol and triglycerides levels were similar among groups. Finally, we report co-localization of EDA
+
-FN with TLR4 on macrophages in human coronary atherosclerotic plaque samples.

Conclusions:
Endogenous EDA
+
-FN exacerbates atherosclerosis via TLR4. Targeting EDA
+
-FN/TLR4 pathway may have the potential to reduce atherosclerosis and improve outcomes in patients at high risk for coronary artery disease.
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 649: PARP14 and PARP9 Are Novel Regulators of Macrophage Activation
Authors: Iwata Hiroshi , Ricchiuto Piero , Hara Takuya , Sharma Amitabh , Mojcher Alex , Fukuda Daiju , Barabási Albert-László , Aikawa Elena , Singh Sasha , Aikawa Masanori
Published: July 2021

Purpose:
A microenvironment dominant in pro-inflammatory macrophages (“M1”) and lacking anti-inflammatory macrophages (“M2”) may promote vascular diseases. We explored and validated key regulators of such macrophage polarization.

Methods and Results:
Using global proteomic analysis and bioinformatics, we examined the changes in the proteomes of mouse and human macrophage cell lines (RAW264.7; THP-1) in response to interferon gamma (IFNγ) or interleukin 4 (IL-4) for M1 or M2 polarization, respectively. Among 5816 proteins in RAW264.7 and 4723 in THP-1, data filtering and clustering identified poly(ADP-ribose) polymerase 14 (PARP14) and 9 (PARP9) as candidates for key regulators of macrophage polarization, which increase in M1 and decrease in M2 condition. siRNA silencing of PARP14 in macrophages induced M1 genes TNF-α, IL-1β and iNOS, while decreased M2 markers Arg1 and MRC1, indicating that PARP14 suppresses pro-inflammatory macrophage activation and promotes anti-inflammatory polarization. PARP14 silencing induced STAT1 phosphorylation and reduced STAT6 phosphorylation, suggesting their roles in the underlying signaling mechanisms. In contrast, PARP9 silencing decreased M1 markers, as well as phosphorylation of STAT1. Of interest, a direct physical interaction between PARP14 and PARP9 was also demonstrated. In vivo evidence supported these in vitro findings. Macrophages of PARP14-deficient mice expressed markedly higher levels of M1 genes and lower levels M2 markers. PARP14 deficiency accelerated lesion development after mechanical injury in femoral arteries.

Conclusions:
PARP14 and PARP9 regulate macrophage activation, offering novel therapeutic targets for vascular diseases.
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 656: N-acetylcystein Reduces Lipid Peroxidation and Advanced Glycation Related to Prevention of Macrophage Endoplasmic Reticulum Stress Induced by Albumin Isolated from Rats With Chronic Kidney Disease
Authors: Machado Juliana T , Iborra Rodrigo T , Fusco Fernanda B , Castilho Gabriela , Pinto Raphael S , Machado-Lima Adriana , Nakandakare Edna R , Shimizu Maria Heloisa M , Seguro Antonio Carlos , Catanozi Sergio , Passarelli Marisa
Published: July 2021

Objective:
We analyzed the influence of N-acetylcysteine (NAC) in chronic kidney disease (CKD) rats on the plasma concentration of lipid peroxides (TBARS) and advanced glycation end products (AGE) and on the impact of serum CKD-albumin in the development of macrophage endoplasmic reticulum stress (ERS).

Methods:
CKD was induced by 5/6 nephrectomy in 2-month old male Wistar rats. Controls (C) were sham operated. Animals were treated or not with NAC (600mg/L of water). FPLC isolated serum albumin was purified by alchoolic extraction. J774 macrophages were incubated with serum albumin (1mg/mL; 18h) from all groups, and the expression of ERS markers (protein disulfide isomerase - PDI and Grp94 chaperone) determined by immunoblot. Comparisons were done by one-way ANOVA, Student t test.

Results:
After 60 days of CKD, body weight was 10% lower in CKD compared to C (p<0.01). This was prevented by NAC. Urea, creatinine, total cholesterol (TC), triglycerides (TG) (mg/dL), urinary protein excretion (mg/24h) (C, n= 31; C+NAC, n=20; CKD, n=74; CKD+NAC, n=32), total AGE and pentosidine (n= 8; fluorescence arbitrary unit) and TBARS (n= 7; nmoL/mL) were higher in CKD (122±8; 0.9±0.07; 151±6; 83±4; 46±2.5; 32620±673; 16700±1370; 6.6±0.5, respectively) and in CKD+NAC (91.4±5; 0.6±0.02; 126±7.5; 73±6; 51±3.5; 24,720±1,114; 10,080±748; 4.5±0.5, respectively) in comparison to C (41±0.9; 0.4±0.03; 76±2.7; 51.5±3; 14±0.9; 21,750±960; 5,314±129; 2±0.2, respectively; p<0.001) and C+NAC (40±0.9; 0.3±0.02; 76±2.6; 68±4; 18.4±1.5; 20,040±700; 5,050±267; 1.8±0.2, respectively; p<0,001). TC, urea, creatinine, total AGE, pentosidine and TBARS were respectively, 17%, 25%, 33%, 24%, 40% and 28% (p<0.01) lower in CKD+NAC, than in CKD. Glycemia was higher in C+NAC (107±4.6) and CKD+NAC (107±2.6) than in C (96±1.8; p<0.05) and CKD (98±1.6; p<0.01), respectively. In macrophages (n=6), CKD albumin increased PDI (5 and 7 times, p<0.01) and Grp94 (66% and 80%, p<0.01) in comparison to C and CKD+NAC-albumin treated cells, respectively.

Conclusion:
NAC reduces plasma lipid peroxidation and AGE and abrogates ERS induced by CKD-albumin. This may contribute to attenuate the deleterious effects of CKD-albumin on lipid accumulation in macrophages helping preventing atherogenesis in CKD.
Abstract 657: A New Translational Model of Hypercholesterolemia and Atherosclerosis: Effect of Statins on LDLR KO Miniature Swine
Authors: Mais Dale E , Vihtelic Thomas , Amuzie Chidozie , Denham Steven , Swart John R , Rogers Christopher S
Published: July 2021

Small animal models of atherosclerosis are commonly used in drug studies; however, the results often fail to translate into the clinic. A large animal model that more accurately reflects the human disease is needed. We recently developed a transgenic Yucatan pig model in which the LDL receptor (LDLR) gene is knocked out. Five groups of Yucatan pigs (N=4 per group), either wild type (LDLR+/+) or heterozygote (LDLR+/-) were fed a normal diet or a high fat diet for a six month period. One of the heterozygote/high fat diet groups in addition received a daily dose of a statin (atorvastatin) at 3 mg/kg. Every two weeks during the study a variety of clinical chemistry parameters were measured. At study termination, select arteries were collected, stained for lipid deposits and quantitated. In addition, sections of these arteries were prepared for immunohistochemistry to detect selected markers of macrophage infiltration into the atherosclerotic plaques. As expected, pigs fed a high fat diet gained significantly more weight at six months whether they were wild type or LDLR+/-. Atorvastatin appeared to attenuate this weight gain. There were significant increases in total cholesterol, HDL and LDL in pigs fed the high fat diet compared to their corresponding control group. The group receiving the atorvastatin had reduced values of these parameters compared to controls showing that a statin had a beneficial effect on lipid levels even in a high fat diet scenario. VLDL levels were not affected but there were triglyceride changes across the groups. Liver function was unchanged based on total bilirubin and AST while ALT measurements were altered in some of the groups. Immunohistochemistry and histomorphometry was performed on some arteries. Atorvastatin-induced amelioration of hypercholesterolemia in this model underscores its translational utility.
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 67: Intervention With Citrus Flavonoids Reverses Existing Metabolic Disorders and Attenuates the Progression of Advanced Atherosclerosis in High Fat--Fed LDLr-/- Mice
Authors: Burke Amy C , Sutherland Brian G , Sawyez Cynthia G , Telford Dawn E , Umoh Joseph , Drangova Maria , Huff Murray W
Published: July 2021

Previous studies demonstrated that addition of the citrus flavonoids naringenin or nobiletin to a high-fat diet prevented the development of many disorders linked to the metabolic syndrome. In the present study, we assessed the ability of intervention with nobiletin or naringenin to reverse pre-established obesity, insulin resistance, hepatic steatosis, dyslipidemia and attenuate atherogenesis. Ldlr-/- mice were fed chow or a high-fat, cholesterol-containing (HFHC) diet for 12 weeks. For an additional 12 weeks, the HFHC-fed mice: (1) continued on the HFHC diet or were transferred to (2) chow, (3) HFHC + 3% naringenin, or (4) HFHC + 0.3% nobiletin. Following rapid weight gain during HFHC-induction, intervention with naringenin or nobiletin stimulated weight loss, while maintaining caloric intake. Micro-CT imaging revealed flavonoid intervention reversed adipose tissue accumulation by 40-60% in both subcutaneous and visceral depots. At 12 weeks, the HFHC-fed mice were hyperinsulinemic (6-fold), which was accompanied by increased fasting plasma glucose. Intervention with either flavonoid normalized plasma insulin and glucose and corrected impaired insulin and glucose tolerance. The HFHC diet increased cholesterol within VLDL (10-fold) and LDL (6-fold), which was reduced (~50%) by either naringenin or nobiletin intervention. HFHC-induction significantly increased hepatic steatosis. Flavonoid intervention reduced hepatic cholesterol (>50%) and triglyceride (~20%) via increased expression of Pgc1a and Cpt1a and reduced expression of Srebp1c. HFHC-induction increased atherosclerotic lesion area (13-fold), which was increased a further 2.5-fold at 24 weeks. Flavonoid intervention modestly retarded lesion size progression (16-20%). As well, intervention with naringenin or nobiletin slowed the accumulation of aortic cholesterol (~30-45%) and reduced lesional necrotic area (~25%), suggesting improved lesion morphology. These studies demonstrate in mice with pre-existing metabolic dysregulation and atherosclerosis that intervention with naringenin or nobiletin reverses obesity, dyslipidemia, hepatic steatosis and insulin resistance, and modestly attenuates the progression of advanced atherosclerosis.
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 78: Extracellular RNA Induces Venous Thrombosis Through TLR3
Authors: Lemarie Catherine A , Le Angela N , Laurance Sandrine , Bertin Francois-Rene , Blostein Mark D
Published: July 2021

Venous thromboembolism (VTE) afflicts 117 people per 100,000 each year and is an important cause of morbidity and mortality. Vascular and circulating cells may sense ‘pathologic ligands’ accumulating in the circulation via toll-like receptor (TLR) and translate signals into a pro-thrombotic state. TLR3 recognizes self-molecules generated during tissue damage and inflammation such as extracellular RNA (eRNA). We hypothesize that eRNA and TLR3 are involved in the development of venous thrombosis after vessel injury. Injection of RNase1 decreased the size of thrombi after FeCl3-induced inferior vena cava injury (IVC) compared to mice treated with vehicle. Using a specific fluorescent probe for RNA, we found that FeCl3 induced RNA release and thus increased RNA content in the thrombus. WT or TLR3 deficient (-/-) mice received an injection of vehicle, eRNA or poly(I:C) prior to thrombosis induction. In WT mice, thrombus size was increased by eRNA or poly(I:C). Interestingly, no change in thrombus size was observed in TLR3-/- mice regardless of the treatment. Poly(I:C) treatment was associated with higher amount of neutrophils within the thrombus of WT mice compared with vehicle. In TLR3-/- mice, poly(I:C) failed to increase the recruitment of neutrophils. Staining for citrulinated H3, a marker of neutrophil extracellular traps (NETs) formation, was increased by poly(I:C) in WT mice. Interestingly, in TLR3-/- mice, no increase of NET formation was found after poly(I:C) injection as compared to vehicle control. Poly(I:C) also increased the recruitment of monocytes within thrombi in WT but not in TLR3-/- mice. Preliminary results suggest that injection of eRNA may also promote the recruitment of monocytes, neutrophils and the formation of NETs in WT but not in TLR3-/- mice. Human neutrophils were then treated with conditioned media from HUVEC treated with poly(I:C) in presence of control siRNA or TLR3 siRNA. Media from HUVEC transfected with control siRNA and treated with poly(I:C) induced the migration of neutrophils, whereas media from TLR3 siRNA and poly(I:C) treated cells did not. These results suggest that eRNA and TLR3 may participate to thrombus formation by inducing a pro-inflammatory response leading to the recruitment of monocytes and neutrophils.
Abstract 84: Deletion of the Type II TGF-ß Receptor in Smooth Muscle Causes Severe Aortopathy in Mice
Authors: Hu Jie Hong , Jaffe Mia , Du Liang , Yan James , Angelov Stoyan , Noh John , Dichek David
Published: July 2021

Introduction:
Prenatal deletion of the type II TGF-beta receptor (TBRII) prevents normal vascular morphogenesis, impairs smooth muscle cell (SMC) differentiation and matrix accumulation, and results in embryonic death in mid-gestation (~embryonic day 13-14). However, the role of TBRII in adult SMC is unknown. Clarification of the postnatal role of TBRII has important clinical implications because TBRII deletion should ablate TGF-beta signaling and blockade of TGF-beta signaling is currently envisioned as a treatment for human aortopathies.

Hypothesis:
We hypothesized that postnatal deletion of TBRII in SMC would impair aortic homeostasis, resulting in aortopathy.

Methods:
We bred mice with a tamoxifen-inducible SMC-specific Cre allele and a floxed Tgfbr2 allele. Mice (6-19 wk old) were injected with tamoxifen or vehicle, and euthanized 4-14 wk later. Aortic mRNA and protein were extracted and Tgfbr2 mRNA transcripts and TBRII protein measured. Aortas were examined grossly after a thoracoabdominal incision, and a subset was examined histologically using both H&E and VVG stains.

Results:
There were no sudden deaths. Tamoxifen-treated doubly transgenic mice had a ~85% decrease in aortic Tgfbr2 transcripts and decreased TBRII protein on western blot. Tamoxifen-treated doubly transgenic mice exhibited several aortic pathologies, none of which were present either in vehicle-treated transgenic or tamoxifen-treated nontransgenic mice: aortic intramural hematoma (19 of 69), rare frank aortic dissection with blood in a large false lumen, penetrating aortic ulcers (7 of 9), medial thickening (46% increase; P < 0.001), aortic dilation, and adventitial thickening. Most of these pathologies were confined to the ascending aorta and arch. In descending thoracic aortas we observed occasional dilation (grossly) and consistent adventitial thickening (on histologic sections). No definite pathology was observed in abdominal aortas.

Conclusions:
TBRII expression in SMC plays a critical role in maintaining postnatal aortic homeostasis. Because loss of TBRII disrupts TGF-beta signaling, these results suggest that blockade of TGF-beta signaling in humans may cause aortic disease rather than prevent it.
Abstract 102: Cd98 Modulates Atherosclerotic Plaque Morphology by Regulating Smooth Muscle Cell Proliferation via the P-38 Map Kinase Pathway
Authors: McCurdy Sara , Baumer Yvonne , Hess Franz , Boisvert William A
Published: July 2021

Smooth muscle cells (SMC) are known to migrate and proliferate to form a stabilizing fibrous cap that encapsulates atherosclerotic plaques. It has been shown that CD98hc, a transmembrane protein with a known role in amino acid transport and integrin signaling, is involved in proliferation and survival of various cell types including SMC. Based on these data, we hypothesized that CD98hc deficiency selectively in SMC would have pathogenic effects on atherosclerosis development and plaque composition. To test this, we utilized mice with SMC-specific deletion of the CD98hc (

CD98hc
fl/fl
SM22Cre
+
)

to determine the effects of CD98hc deficiency on SMC function in the context of atherosclerosis. We performed
in vitro
proliferation and survival/apoptosis assays to investigate the role of CD98hc in the proliferation and survival of primary mouse aortic vascular smooth muscle cells. We found that VSMC isolated from whole aortas of CD98hc
-/-
animals displayed approximately 60% reduced cell counts compared to control (41 ± 8.2% of control) after 5 days in culture. EdU assays
in vivo
showed a defect in the ability of CD98hc
-/-
SMC to proliferate, with 25% reduction in EdU-positive VSMC compared to controls (2.3 ± 0.2% vs 3 ± 0.2%). In addition, caspase-3 staining of SMC
in vitro
displayed a 41% increase in propensity of CD98hc
-/-
SMC to undergo apoptosis compared to controls (7.9 ± 0.6% vs 5.6 ± 0.5%). Furthermore, the absence of CD98hc in SMC caused a sharp increase in phosphorylated p-38, which was partially abrogated towards control levels when the cells were treated with PDGF-BB to induce proliferation. Long-term atherosclerosis study using SMC-CD98hc
-/-
/LDLR
-/-
mice showed that atherosclerotic plaque morphology was altered with increased necrotic core area (25.8 ± 1.9% vs 10.9 ± 1.6% necrotic core area per plaque area) due to a reduction in infiltration of SMC within the plaque (2.1 ± 0.4% vs 4.3 ± 0.4% SM22α positive area per plaque area) compared to control LDLR
-/-
mice. These data support an important role for CD98hc and its regulation of p-38 MAP kinase signaling in aortic vascular smooth muscle cell proliferation and survival. We conclude that CD98hc is critical for the formation of fibrous cap that is important in maintaining the stability of atherosclerotic plaque.
Abstract 113: Phospholipid Physical State Determines the Efficiency of Cholesteryl Ester Transfer by Cholesteryl Ester Transfer Protein between Discoidal High Density Lipoproteins
Authors: Dergunov Alexander D , Litvinov Dmitry Y , Vlasov Mikhail S
Published: July 2021

The transfer of cholesteryl ester by recombinant cholesteryl ester transfer protein (CETP) between reconstituted discoidal HDL was studied. Particles contained apolipoprotein A-I, unsaturated palmitoyllinoleoylphosphatidylcholine (PLPC), palmitoyloleoylphosphatidylcholine (POPC) or saturated dipalmitoylphosphatidylcholine (DPPC) and cholesteryl ester up to 3 mol% as cholesteryl 1-pyrenedecanoate (CPD) or cholesteryl laurate (CL) in donor and acceptor rHDL, respectively. The concentration dependencies of excimerization upward deviated from a linear dependence, being more curvilinear for DPPC. Separately for DPPC complexes, monomer fluorescence compared to excimer was more efficiently quenched by acrylamide. The nonradiative energy transfer between apoA-I tryptophan residues and CPD molecules was more efficient for PLPC compared to DPPC complexes. Data fulfilled the quenching sphere-of-action model, the sphere with adjacent probe molecules being larger and more distant from apoA-I tryptophan residues in DPPC complexes. CETP-catalyzed cholesteryl ester exchange between donor and acceptor HDL followed by probe excimerization was characterized by a heterogeneous kinetics; the fast exchanging CPD pool was much higher in a case of POPC compared to DPPC complexes. Probe fraction accessible to CETP increased with temperature, suggesting a more homogeneous probe distribution. The noncompetitive inhibition of probe transfer by acceptor particles seems to satisfy the zero off-rate compatible with probe tunneling. The values of
V
max
(0.063 μM·min
-1
) and
k
cat
(0.42 s
-1
) together with a similarity of
K
m
(0.9 μM CPD) and
K
I
(2.8 μM CL) values for POPC-containing rHDL suggest the efficient cholesteryl ester transfer between nascent HDL with unsaturated phosphatidylcholine
in vivo
opposite to much less efficiency with HDL with saturated phosphatidylcholine. The physical state of phospholipid matrix may underlie CETP activity with discoidal HDL through self-association and location of cholesteryl ester in the bilayer, the availability of cholesteryl ester to cholesterol-binding site in apoA-I structure and the binding of apoA-I-interacting cholesteryl ester to CETP. We are grateful to Dr. A. Tall for providing the recombinant CETP.
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 126: Effects of Rosuvastatin on the Protein Composition of Lipoprotein Subfractions
Authors: Gordon Scott M , Kemeh Georgina , Fessler Michael B , Remaley Alan T
Published: July 2021

Introduction:
Statins, by inhibiting HMG-CoA reductase and up regulating hepatic LDL receptors, effectively lower plasma LDL-C by as much as 50%, thus reducing future CVD events. However, the physiological effects of statins are diverse and not all are related to lowering of LDL-C. Goal: The goal of this study was to test our hypothesis that some of these pleiotropic alternative effects from statins may be driven by compositional changes to lipoproteins distinct from their cholesterol content. We, therefore, performed a small clinical pilot study to assess the impact of statins on lipoprotein associated proteins in healthy individuals.

Methods:
Ten subjects with normal LDL-C (<130 mg/dL) were given rosuvastatin (20 mg/day) for 28 days. Plasma samples collected at baseline and after treatment were used for lipid measurement, nuclear magnetic resonance (NMR) lipoprotein profiling and lipoprotein proteomics.

Results:
The effects of rosuvastatin treatment on clinical lipid measures and NMR profile were consistent with established findings. Proteomic analysis of FPLC fractions representing LDL, HDL-1 (large) and HDL-2 (small) identified a total of 124 different proteins. Spectral counting was used to compare relative protein detection before and after statin therapy. Significant protein changes were found in each lipoprotein pool: LDL = 9, HDL-1 = 9 and HDL-2 = 4. These changes included both increases and decreases in proteins involved in lipoprotein metabolism, complement regulation and acute phase response. The most dramatic effect of the treatment was a profound increase in alpha-1-antirypsin (A1AT) spectral counts association with HDL-1 particles. Quantitative measurement by ELISA revealed an average 5.7 fold increase in HDL-1 associated A1AT. Preliminary in vitro studies indicate a potential functional role for A1AT enriched HDL in the formation of neutrophil extracellular traps (NETs), a pro-inflammatory component of vascular lesions.

Summary:
Based on these results, statins can significantly change the protein composition of both LDL and HDL. Some of these changes, such as the up regulation of A1AT on HDL, may convey anti-inflammatory functionality on lipoproteins and might contribute to some of the non-lipid lowering effects of statins.