bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 185: Using the Platelet Proteome to Identify Novel Biomarkers in Heart Failure With Preserved Ejection Fraction
Authors: Purushotham Diana , Raphael Roseanne , Gastonguay Courtney , Chesnik Marla , Mirza Shama , Strande Jennifer L
Published: July 2021

Introduction:
The platelet proteome is an untapped resource for identifying novel proteins that may reflect a disease process. The platelet proteome is understudied in heart failure. We hypothesize that the platelet proteome will change composition in patients with heart failure with ejection fraction (HFpEF). Using mass-spectral analysis, we examined the platelet proteome from subjects with heart failure with preserved ejection fraction (HFpEF) to identify proteins associated with the disease process.

Methods/Results:
We conducted a case-controlled prospective study in which we drew blood samples from hospitalized subjects with symptoms of HFpEF (n=9), the same subjects several weeks later without symptoms (n=7) and control subjects (n=8). The subjects with HFpEF were older and had a higher incidence of atrial fibrillation and smoking compared to the control group. There were no significant differences in body mass index, and history of hypertension, hyperlipidemia, coronary artery disease or diabetes. The HFpEF group had increased LV wall thickness, left atrial volume, and diastolic dysfunction. Mass spectrometry of the platelet rich abstract obtained from the blood samples of the 3 groups identified 6,102 proteins with 5 scans with peptide probabilities of ≥0.85. Of the 6,102 proteins, 165 were present only in symptomatic subjects, 78 were only found in asymptomatic subjects and 157 proteins were unique to the control group. S100A8 and its receptor, CD36 were two proteins identified consistently in HFpEF samples when compared with controls. Using ELISA, we further confirmed that plasma S100A8 levels were increased in subjects with HFpEF (728±358) vs. control (314±154).

Conclusion:
Platelets may harbor proteins associated with HFpEF. S100A8 is present in the platelets of subjects with HFpEF and increased in the plasma of the same subjects. S100A8 has been linked with other cardiovascular disease such as atherosclerosis and risk for myocardial infarction, stroke, or death. This is the first report on the association between S100A8 and HFpEF.
Abstract 2: Ogg1 Plays A Protective Role In Diet Induced Atherosclerosis in LDLR KO mice.
Authors: Tumurkhuu Gantsetseg , Shimada Kenichi , Crother Timothy R , Zhang Wenxuan , Gottlieb Roberta , Chen Shuang , Arditi Moshe
Published: July 2021

Introduction:
In human atherosclerosis, mitochondrial DNA (mtDNA) damage occurs in both circulating cells and the vessel wall, suggesting the importance of DNA damage repair enzymes. Mitochondria are a central regulator of NLRP3 function, which is a sensor of specific pathogen, host, and environmental danger molecules. Oxidized mtDNA can bind to and activate the NLRP3 inflammasome, which cleaves and activates interleukin-1β (IL-1β) and IL-18, cytokines that are pro-atherogenic. Ogg1, an oxoguanine glycosylase, can repair oxidized DNA.

Objective:
To assess the importance of the oxidative DNA damage repair gene, Ogg1, in atherogenesis.

Methods and Results:
To investigate the role of Ogg1 in atherogenesis, we generated Ogg1-/-Ldlr-/- mice. These mice had significantly larger plaques and greater lipid content in the aorta and the aortic root. Elevated levels of 8-OH-dG (a marker of oxidative DNA damage), Caspase-1 activation by FLICA staining, and apoptosis by TUNEL staining, were increased in Ogg1-/-Ldlr-/- animals compared with Ldlr-/- controls. Despite similar concentrations of plasma cholesterol, triglyceride, lipoprotein profile, glucose, insulin resistance, and body weight gain after 16 weeks high fat diet, serum MCP-1 was higher in Ogg1-/-Ldlr-/- mice compared with Ldlr-/- mice. Transplantation with Ogg1-/- bone marrow (BM) into irradiated recipient Ldlr-/- mice also resulted in an increase in atherosclerotic lesion size, 8-OH-dG accumulation, and IL-1β production in aortic tissue compared with control chimeric mice. However, Ogg1-/-/Nlrp3-/- BM transplanted into Ldlr-/- mice did not lead to acceleration of atherogenesis, suggesting that Ogg1 deficiency-induced acceleration of atherosclerosis was mediated by the NLRP3 inflammasome. Indeed, analysis of Ogg1-/- macrophages found a significant increase in caspase-1 activation and IL-1β secretion in these cells. Thus Ogg1 activity is anti-atherogenic.

Conclusions:
Our study provides direct evidence of a link between mtDNA damage, DNA damage repair enzyme OGG1, and atherosclerosis through increased inflammation via the NLRP3 inflammasome. These new data suggest that therapeutics designed to limit mtDNA damage may provide protection from atherosclerotic lesion development.
Abstract 201: A New Multimodal Contrast Agent for Molecular Imaging of Adhesion Molecules Expressed in Atherosclerosis
Authors: Ahmed Mona , Gustafsson Björn , Caidahl Kenneth
Published: July 2021

Background:
Inflammation is involved in many chronic diseases, one of which is atherosclerosis. Early detection of inflammation could improve clinical management. Most existing contrast agents on the market are non-specific and single-modal. Our aim was to apply an innovative new multimodal contrast agent, a polyvinyl alcohol based microbubble (MB), to target adhesion molecules expressed in atherosclerosis, opening a new scenario for biomedicine.

Objective:
To evaluate a contrast agent with different surface modifications and their adhesion to inflammatory
in vitro
models as a proof of concept, by imaging inflamed endothelium, that is involved in the pathogenesis of atherosclerosis.

Methods:
Microbubbles were antibody (Ab) or peptide conjugated to the adhesion molecule of interest. To evaluate attachment between MBs and an inflamed murine cell line, endothelial cells (EC), confocal laser scanning microscopy (CLSM) and flow cytometry (FACS) were used. To verify expression of cell adhesion molecules we used FACS and western blot. Stimulated and non-stimulated cells were incubated with MBs conjugated with active ligands, or a negative control.

Results:
In endothelial cells CLSM showed an increased uptake, or adherence, of peptide-conjugated microbubbles compared to non-stimulated control. MBs tagged with Abs against; ICAM-1, VCAM-1, E-Selectin, or with Cyclic Arginine-Glycine-Aspartate (cRGD) peptide showed stronger adherence to EC compared to non-targeted control. Microbubbles labeled with cRGD-peptide was shown to be the most prominent, indicating a specific interaction.

Conclusions:
Adhesion molecules expressed in atherosclerosis may be targeted by our labeled multimodal contrast agent
in vitro
, with potential for disease specific diagnostics
in vivo.
Abstract 212: Role of Monocytes and Inflammation in Vascular Endothelial Dysfunction in Mice With Heart Failure After Myocardial Infarction
Authors: Molitor Michael , Finger Stefanie , Kossmann Sabine , Garlapati Venkata S , Lagrange Jérémy , Schüler Rebecca , Münzel Thomas , Karbach Susanne , Knorr Maike , Wenzel Philip
Published: July 2021

Background:
Heart failure (HF) after myocardial infarction (MI) leads to impaired left ventricular function and reduced blood flow in peripheral arteries. Angiotensin II (ATII) signaling is crucial in MI, and inflammatory myelomonocytic cells are involved in the process of ATII-induced vascular dysfunction. Their role in MI-mediated vascular dysfunction has not been defined yet.

Objective:
Test the impact of selective depletion of lysozyme M positive (LysM+) myelomonocytic cells on endothelial dysfunction in a model of ischemic heart failure in mice

Methods and results:
8 to 12 week old mice (C57B6 background) were subjected to permanent coronary ligation of the left anterior descending artery (LAD) to induced HF post MI. We measured a reduced vascular endothelial and smooth muscle function in isolated aortic segments 7d and 28d post MI in isometric tension studies. Also the production of vascular superoxide (chemiluminescence, oxidative fluorescence microtopography) and vascular mRNA expression of MCP-1, VCAM-1, IL-6 and ATII receptor type 1 were increased (assessed by mRNA reverse transcription polymerase chain reaction) in HF mice compared to sham. FACS analysis of aortic tissue of HF mice shows an increased infiltration of CD45+ immune cells in the aortic wall, including CD11b+Gr-1lowF4/80+ monocytes/macrophages. Using mice with LysM dependent cre-inducible expression of diphtheria toxin receptor (LysMCreIDTR) we selectively ablated LysM+ myelomonocytic cells 28d after MI for 10d via diphtheria toxin. We assessed a significantly improved vascular endothelial and smooth muscle function, less vascular superoxide production and a reduced expression of VCAM-1 and ATII receptor type 1 in aortic tissue. The number of circulating monocytes in blood was reduced, while the cardiac function (EF (%), LV size) in sonography stays constant between depleted and non-depleted HF mice.

Conclusion:
Our results suggest that vascular dysfunction post MI is at least in part mediated by inflammatory leukocyte infiltrating the vessel wall and that depletion of inflammatory monocytes in HF after MI improves the vascular function. It can potentially be a new target to protect endothelial function in HF and prevent secondary events after a MI.
Abstract 224: Detoxification versus Healthy Aging: Aryl Hydrocarbon Receptor Deficiency Improves Vessel Functionality and Increases Healthy Lifespan
Authors: Altschmied Joachim , Eckers Anna , Jakob Sascha , Heiss Christian , Goy Christine , Brinkmann Vanessa , Ventura Natascia , Haendeler Judith
Published: July 2021

Development of age-associated vascular diseases like atherosclerosis depends not only on genetic predisposition but also on environmental influences. Ligands of the aryl hydrocarbon receptor (AhR), a ubiquitously expressed transcription factor upregulating detoxifying enzymes, like dioxin and benzo[a]pyrene (BaP) have been shown to promote atherosclerosis. Furthermore, recovery of the blood flow after hindlimb ischemia is significantly enhanced in AhR-deficient mice demonstrating increased angiogenesis in the absence of AhR. Thus, there seems to be a link between AhR, vessel functionality and age-related cardiovascular diseases.

To investigate the role of the AhR in health span and vessel function, we analyzed AhR-deficient Caenorhabditis elegans, vessel stiffness in AhR-knockout mice and human subjects of different age and with varying levels of AhR expression as well as functional parameters in primary human endothelial cells (EC) after AhR activation.

AhR-deficient C. elegans showed not only an extended life span, but also enhanced motility. In AhR-knockout mice, we observed a reduced PWV in both old and young animals, suggesting that AhR impairs vessel function already at young age. Concomitantly, eNOS phosphorylation at serine 1178, a surrogate marker for eNOS activation, was enhanced in aortas of knockout animals. In line with this, the AhR agonist BaP increased an inhibitory phosphorylation on eNOS in EC. Moreover, BaP reduced migration of EC without changes in proliferation or apoptosis, an effect that was reversed by addition of the AhR antagonist 3’methoxy-4’nitroflavone. In human subjects we demonstrated not only a positive correlation between age and pulse wave velocity (PWV), a readout for vessel stiffness, but also between AhR expression in blood cells and PWV, again suggesting a negative impact of AhR on vessel functionality.

Our data demonstrate that loss of AhR extends life span as well as health span in C. elegans. Knockout of AhR in mice leads to improvement of vessel functionality by decreasing vessel stiffness. Finally, the PWV in humans positively correlates not only with age but also with the expression of AhR. Thus, AhR expression may be useful as a new predictor of healthy aging from nematodes to humans.
Abstract 225: Parallel and Divergent Patterns of Gene Expression in Human Arteriovenous Fistula Maturation and Stenosis
Authors: Benrashid Ehsan , Yerxa John , Lucas Kathryn A , Gabr Mostafa A , Haley Jennifer , Serrano Sabrina , McCoy Christopher C , Otto James C , Lawson Jeffrey H
Published: July 2021

Background:
The biologic mechanisms that differentiate successful arteriovenous fistula (AVF) maturation from stenosis are not well understood. The brachiobasilic transposition offers an opportunity for studying the vascular remodeling process. This procedure is a two stage process: first to create a brachiobasilic AVF and second, to transpose the AVF near the skin in a delayed fashion, allowing the opportunity to harvest naïve vein, mature and stenotic AVF tissue. Identifying the molecular differences with regards to success (maturation) or failure (stenosis) are vital both for dialysis access care and as a model of vein remodeling on exposure to arterial flow. We hypothesize that basilic vein transposition (BVT) can be utilized as a unique means with which to study the biologic factors that affect stenosis and maturation.

Methods/Results:
Vein tissue was obtained at the time of initial access creation as naïve segments and again at the time of basilic vein transposition (following venous “arterialization”), resulting in a cohort of patient-matched naïve, mature, and stenotic segments. Time between procedures ranged from 3-8 months, with a mean of 5. Vein segments were scored at time of collection as mature or stenotic, and confirmed by histology. Real-time PCR was performed on extracted RNA using a panel of genes implicated in the process and regulation of both successful and “unsuccessful” vascular wall remodeling. Heme oxygenase-1, matrix metalloproteinase-9 (MMP9) and osteopontin (SPP1) are significantly upregulated in both mature and stenotic vein segments (vs. naïve); MMP9 and SPP1 were also significantly upregulated in mature vs. stenotic tissue. SPP1 was exclusively expressed in the medial region of mature and stenotic AVF. Stenotic samples demonstrated a significant increase in the expression of tissue inhibitor of metalloproteinase-1 (MMP-1) and endothelin-1 (EDN-1) versus both naïve and mature samples.

Conclusion:
The BVT offers a unique opportunity to identify potential mechanisms involved in AVF maturation and stenosis. There appears to be overlap in gene expression profiles for both mature and stenotic samples, however MMP-1 and EDN-1 appear to be significantly increased in venous stenosis.
Abstract 266: N-Acetyl-Cysteine Can Alter the Endothelial Cell Lipid Microenvironment
Authors: Zayed Mohamed A , Wei Xiachao , Belaygorod Larisa , Hsu Fong-Fu , Semenkovich Clay
Published: July 2021

The endothelial cell (EC) lipid microenvironment can directly impact post-translational lipid modification of critical intracellular signaling proteins, which has important vascular disease implications. The antioxidant N-Acetyl-Cysteine (NAC) is a well-known thiol that regulates intracellular glutathione levels, activates various intracellular kinases, and promotes EC proliferation. In erythrocytes, NAC alters cellular lipid content and modulates generation of plasma membrane phosphatidylserine (PS). To test the hypothesis that NAC similarly alters the lipid microenvironment in ECs, we evaluated the effect of NAC on S(cysteine)-protein lipidation, as well as its effects on cellular lipid content in HUVECs. Increasing the duration of NAC treatment (from 2 to 16 hours) and increasing the concentration of NAC (from 1 to 5 mM) robustly decreased S-protein lipidation of the heterotrimeric G protein subunit, Gaq, by 80% (p<0.05) without affecting Gaq protein levels. There was no effect of NAC on lipidation of the Caveolin-1, a lipid raft protein known to be involved in vascular function. NAC also suppressed expression (by 55%, p<0.05) of Fatty Acid Synthase (FAS), a critical lipogenic enzyme implicated in generating palmitate utilized for the lipidation of vascular proteins. Since Gaq-mediated regulation of downstream phospholipases affects cytosolic phospholipids, we characterized lipidomic profiles of NAC-treated ECs using electrospray ionization mass spectrometry analysis. Preliminary findings demonstrated that treatment with NAC 5mM alters global PS and phosphatidylcholine (PC) content in HUVECs, with a 160% increase in total PS lipid species and a 140% increase in total PC lipid species. These findings suggest that NAC remodels the lipid milieu of endothelial cells in a manner likely to impact signaling pathways relevant to inflammation, proliferation, and repair in the vasculature.
Abstract 280: Neointimal Hyperplasia in Allogeneic and Autologous Venous Grafts Appears Homogenous
Authors: Busch Albert , Hartmann Elena , Wagner Nicole , Ergün Süleyman , Kickuth Ralph , Kellersmann Richard , Maegdefessel Lars , Lorenz Udo
Published: July 2021

Introduction:
Cryopreserved venous allografts are considered as possible alternative to autologous venous grafts in peripheral bypass surgery. Neointima formation is responsible for poorer patency. For a better understanding of this process we investigated allogeneic and autologous venous grafts in arterial position by histologic means.

Materials and Methods:
Allogeneic and autologous venous graft stenosis were examined by Ki67, CD31, CD34, CD3, CD20, vWF, CD68, actin, vimentin and desmin.

Results:
The morphological Examination of autologous and cryopreserved allogeneic grafts appears very uniform. Dedifferentiated vascular smooth muscle cells are the main cellular effector in both grafts. Neoangiogenesis is abundant in the vessel media whereas immunologic infiltrates are largely absent.

Conclusion:
The uniform morphological appearance of allogeneic and autologous neointimal hyperplasia suggests an overreaction in venous grafts in response to non-immunological factors as reason for poorer clinical outcome of cryopreserved allografts.
Abstract 294: Inhibiting Myeloperoxidase Restores Collateralization in Ischemic Hindlimbs in Diabetes
Authors: Weihrauch Dorothee , Krolikowski John G , Jones Deron W , Zhang Hao , Pritchard Kirkwood A
Published: July 2021

Background:
Myeloperoxidase (MPO) is released from activated neutrophils and is believed to play a causal role in peripheral vascular disease (PVD). Leptin receptor deficient (db/db) mice are a model of type 2 diabetes mellitus (DM) that are regularly used to investigate mechanisms by which DM impairs endothelial cell (EC) function and increases PVD.

Objectives and Methods:
To investigate MPO’s role in ischemic disease in DM, db/db mice were pretreated with N-acetyl-lysyltyrosylcysteine amine (KYC, subQ, 1 mg/kd/d) for 7 days, subjected to hindlimb ischemia, treated with KYC for 14 days and changes in blood flow determined by Laser Doppler. Hindlimbs were examined for changes in neutrophil counts, chlorotyrosine (Cl-Tyr), 4-hydroxynoneal (4-HNE), advanced glycation end-products (AGE) expression via immunohistochemistry and immunoblotting. Hindlimb matrix was used to determine the effects of DM and KYC treatments on matrix-dependent EC proliferation and migration.

Results:
Percent change in blood flow in ischemic legs (IL) of C57BL/6J mice increased to ~50% by day 14 post femoral artery ligation compared to day 0. In contrast, blood flow in IL of untreated db/db mice increased to ~13%, ~15% and ~17% on days 3, 7 and 14. Blood flow in IL of db/db mice treated with KYC increased to ~20%, ~43% and ~47% on days 3, 7 and 14. KYC treatments decreased neutrophil counts, Cl-Tyr, 4-HNE and AGE expression in IL. Further, proliferation and migration were increased when EC were cultured on matrix isolated from KYC-treated db/db mice compared to matrix from untreated db/db mice. Interestingly, KYC treatments had no effect on blood glucose in db/db mice.

Conclusions:
MPO increases oxidative stress, tissue injury and impairs EC function to increase PVD in db/db mice. KYC inhibition of MPO reduces oxidative stress and tissue injury and restores EC function and blood flow in db/db mice. These data suggest that KYC may be effective for treating PVD in patients with diabetes.
Abstract 316: Paraoxonase 1 (PON1) Promotes the Cholesterol Efflux and Regulates the Reverse Cholesterol Transport (RCT)
Authors: Ikhlef Souade , Berrougui Hicham , Khalil Abdelouahed
Published: July 2021

Paraoxonase 1 (PON1) is a High Density Lipoprotein (HDL) associated enzyme which contributes to the antioxidant and anti-inflammatory properties of HDL. It also stimulates cholesterol efflux mediated by HDL. The purpose of this study is to determine the mechanism by which PON1 regulates this antiatherogenic activity of HDL. The oxidized phospholipids hydrolysis by PON1 results in the formation of Lysophosphatidylcholine (LPC). The phospholipase PON1 activity effect on the cholesterol efflux and the gene and protein expression were determined in vitro on J774 macrophages cells and was also determined in vivo on C57/BL6 male mice.

Our results show that due to its phospholipase activity, PON1 hydrolyzes oxidized LDL (oxLDL) and contributes to the formation of LPC, which stimulates cholesterol efflux from macrophages. Our results show that PON1 induced the expression of ABCA1 cholesterol transporters. Western blot as well as qPCR analysis show that PON1 stimulate PPARγ and LXRα nuclear receptors expression. Moreover, the use of ABCA1, PPARγ and LXRα inhibitors (respectively DIDS, GGPP and GW9662) affects the capacity of PON1 to stimulate cholesterol efflux, in particular by decreasing the proteins expression of interest (ABCA1, PPARγ and LXRα). Incubation of macrophages with purified LPC with macrophages induces a high expression of PPARγ-LXRα-ABCA1 pathway. Incubation of macrophages with PON1-oxLDL mixture and their injection to mice increase significantly the reverse cholesterol transport. Our results show that tPON1 stimulate both cholesterol efflux and RCT. via the regulation of the PPARγ-LXRα-ABCA1 pathway. This effect is attributed to the phospholipase activity of PON1 which mediates the hydrolysis of ox-LDL to form LPC.
Abstract 324: The Apolipoprotein A-IV T347S Polymorphism is Associated with Severe Hepatic Steatosis in Obese Subjects Undergoing Bariatric Surgery
Authors: Sawyer Janet K , Fernandez Adolfo Z , Rudel Lawrence L , Weinberg Richard B
Published: July 2021

Background:
We recently reported that hepatic apolipoprotein A-IV (apoA-IV) gene expression is increased in mouse models of steatosis and is positively correlated with liver triglyceride (TG) content. The most common human apoA-IV polymorphism T347S (rs675) decreases its lipid affinity and is associated with increased adiposity in population studies.

Hypothesis:
347S allele carriers will exhibit increased hepatic TG content in conditions predisposing to hepatic steatosis.

Methods:
We examined the impact of the 347S allele on liver TG content in a cohort of very obese humans. Total hepatic mRNA was extracted from snap frozen tissue obtained from 40 obese subjects (11 male, 29 female) at the time of bariatric surgery. ApoA-IV abundance was quantitated by RT-PCR; copy numbers were normalized to GAPDH. Presence of the T347S polymorphism and the next most common apoA-IV allele, S127N (rs5104), was determined by cDNA sequencing. Liver total TG was measured in solvent extracts by GLC and normalized as mg TG/g protein.

Results:
Mean BMI in the cohort was 43.8 ± 1.5 kg/m
2
; T347S allele frequency was 0.38 and S127N allele frequency was 0.23; 16/40 (40%) had severe steatosis (hepatic TG content > 400 mg TG/g protein). There was no difference in total hepatic apoA-IV mRNA abundance among carriers of the wild type and variant alleles; mean liver TG content was highest in subjects carrying a 347S allele; a significantly higher percentage of subjects carrying a 347S allele, but not a 127N allele, had severe steatosis (P=0.024).

Conclusions:
These data establish that the apoA-IV 347S allele is associated with severe hepatic steatosis in a cohort of obese subjects, and suggest that apoA-IV genotype plays a role in hepatic lipid metabolism. As apoA-IV facilitates hepatic TG export by enabling secretion of larger VLDL particles, the impact of the 347S allele on hepatic TG content in the absence of altered gene expression suggests that its reduced lipid affinity may impair VLDL particle expansion.
Abstract 327: Liver X Receptors (LXR) Modulate the Negative Effects on Endothelial Progenitors Cells after Feeding a High Cholesterol Diet
Authors: Rasheed Adil , Cummins Carolyn L
Published: July 2021

The liver X receptors (LXRα/β) belong to the nuclear receptor superfamily of ligand activated transcription factors. LXRs are endogenously activated by oxysterols; and one of their primary functions is to facilitate cholesterol efflux out of cells, such as macrophages, via the ABCA1 & ABCG1 transporters. Bone marrow (BM) transplant studies have shown that LXRs are important for preventing the formation of atherosclerotic plaques. Loss of LXRs in the monocyte/macrophage population of the BM was thought to be the primary mediator of the increased atherosclerosis in these studies. However, the BM is comprised of a variety of cell types that are differentiated from hematopoietic stem cells (HSCs). Like numerous other vascular complications, endothelial defects are central to the pathogenesis of the early stages of atherosclerosis. Endothelial progenitor cells (EPCs), derived from HSCs in the BM, contribute to vascular health primarily through the secretion of factors that maintain and repair the endothelium of the damaged vasculature. We hypothesized that LXRs also modulate the negative effects of cholesterol on EPCs, in addition to its effects in macrophages. To study the effects of dietary cholesterol on EPCs and the roles of LXRs in modulating these effects, WT and LXRα/β-/- mice were fed a high-fat/high-cholesterol (HF/HC) diet starting at 8-9 weeks of age for a total of 12 weeks. Complete blood counts showed that in the LXRα/β-/- but not the WT mice fed the HF/HC diet, there was a significant expansion of select myeloid populations (neutrophils, monocytes, platelets). Furthermore, EPCs derived from the HF/HC fed LXRα/β-/- mice had a higher expression of the endothelial markers CD144 and VEGFR2 compared to WT mice and LXRα/β-/- mice on chow diet, suggesting enhanced differentiation to a more endothelial-like cell in the absence of LXRs. Notably, the paracrine reparative actions of EPCs have been shown in populations of cells with low expression of endothelial markers. Taken together, these results suggest that LXRs modulate the effects of HF/HC diet on the differentiation of EPCs to endothelial cells and thus, may provide another cell type within the BM that would benefit from LXR activation in modulating vascular disease.[[Unable to Display Character:  ]]
Abstract 347: Water-soluble Components of Sesame Oil Reduces Inflammation and Atherosclerosis
Authors: Aluganti Narasimhulu Chandrakala , Selvarajan Krithika , Burge Kathryn , Litvinov Dmitry , Sengupta Bhaswati , Parthasarathy Sampath
Published: July 2021

Background:
Atherosclerosis, a major form of cardiovascular disease, has now been recognized as a chronic inflammatory disease. Non-pharmacological means of treating chronic diseases have gained attention recently. We previously reported that the sesame oil aqueous extract (SOAE) has anti-inflammatory properties both in vitro and in vivo. In this study, we have determined whether SOAE has anti-atherosclerotic properties, and mechanisms by which it might modulate atherosclerosis by identifying genes and inflammatory markers.

Methods and results:
Low-density lipoprotein receptor knockout (LDLR-/-) female mice were fed with either high fat diet or high fat diet supplemented with SOAE. Plasma lipids and atherosclerotic lesions were quantified after 3 months of feeding. Plasma samples were used for global cytokine array. RNA was extracted from the liver tissue and the aorta and used for gene analysis. The SOAE-supplemented high fat diet significantly reduced atherosclerotic lesions, plasma cholesterol, and LDL cholesterol levels in LDLR-/- mice. Plasma inflammatory cytokines were reduced, but not significantly, demonstrating an anti-inflammatory property of SOAE. Gene analysis showed that SOAE-supplemented high fat diet reduced the genes involved in inflammation, and induced genes involved in cholesterol metabolism and reverse cholesterol transport.

Conclusion:
In conclusion, our studies suggest that a SOAE-enriched diet could be an effective non-pharmacological treatment for atherosclerosis by controlling inflammation and regulating lipid metabolism.
Abstract 35: Identification of a Mouse Strain Modifier Locus for Factor V Leiden/Tissue Factor Pathway Inhibitor Dependent Thrombosis
Authors: Germain Derrik M , Siebert Amy E , Verbeek Stephanie , Zhu Guojing , Tomberg Kärt , Cleuren Audrey , Siemieniak David , Westrick Randal J
Published: July 2021

Factor V Leiden (FVL) is the most common genetic risk factor for venous thromboembolism (VTE). FVL is incompletely penetrant as only 10% of FVL heterozygotes develop VTE. Previously, nearly uniform perinatal lethality was observed in C57BL/6J (B6) mice hemizygous for tissue factor pathway inhibitor (Tfpi+/-) on a homozygous FVL background (FVQ/Q). However, inbred mouse strains exhibit significant genetic variation and can exert a profound influence on phenotype. We tested the lethality of FVQ/Q Tfpi+/- on the DBA/2J inbred strain background by crossing FVQ/+ Tfpi+/- to DBA/2J followed by a double heterozygous (FVQ/+ Tfpi+/-) x FVQ/Q cross. Surprisingly, FVQ/Q Tfpi+/- mice on the DBA/2J genetic background are born at expected Mendelian frequencies (N=62, nexp=15, nobs=15, N.S.) and display no overt thrombosis. This demonstrates the existence of dominant antithrombotic DBA/2J strain-specific thrombosis modifier gene(s). Analysis of progeny from a DBA/2J-B6 F1 FVQ/Q Tfpi+/- x B6 FVQ/Q cross yielded equal proportions of FVQ/Q Tfpi+/- and FVQ/Q Tfpi+/+ (N=130, nexp=43, nobs=63, p<0.001) which suggested that multiple modifier genes were contributing to the phenotype. To isolate individual dominant modifier loci, we adopted a serial backcrossing strategy in which F1 FVQ/Q Tfpi+/- mice were serially backcrossed to B6 FVQ/Q mice for 17 generations. A standard mouse positional cloning strategy was used on the 110 surviving FVQ/Q Tfpi+/- mice from these crosses which identified a chromosome 2 modifier locus (148-169 Mb) under genetic selection. Currently there are 16 genes in this locus with known annotated non-synonymous coding variants. Ongoing studies are focused on identifying the responsible genetic DBA/2J variant within this locus. Identification of this modifier gene will represent a significant advance for the genetics of thrombotic disease. Not only will it give insight into the pathways leading to thrombosis, it may also reveal previously unexplored therapeutic targets for the prevention of thrombotic events.
Abstract 351: Mechano-sensitive Ppap2b Regulates Endothelial Responses to Athero-relevant Hemodynamic Forces
Authors: Wu Congqing , Kuo Cheng-Hsiang , Kim Chan Woo , Huang Ru-Ting , Birukova Anna , Birukov Konstantin G , Dulin Nickolai O , Civelek Mete , Lusis Aldons J , Dai Guohao , Jo Hanjoong , Fang Yun
Published: July 2021

Rationale:
PhosPhatidic-Acid-Phosphatase-type-2B (PPAP2B), an integral membrane protein that inactivates lysophosphatidic acid, was implicated in coronary artery disease (CAD) by genome-wide association studies (GWAS). However, it is unclear whether GWAS-identified CAD genes including PPAP2B participate in mechanotransduction mechanisms by which vascular endothelia respond to local athero-relevant blood flows that contribute to the regional nature of atherosclerosis.

Approach and Results:
Reduced endothelial PPAP2B was detected in vivo in swine aortic arch exposed to chronic disturbed flow and in mouse carotid artery subjected to surgically-induced acute disturbed flow. In culture, elevated PPAP2B was measured in human aortic endothelial cells (HAEC) under athero-protective flow mimicking hemodynamics of human distal carotid artery when compared with athero-susceptible waveform representing flow in carotid sinus. Disturbed flow-induced miR-92a was identified as a direct posttranscriptional inhibitor of mechano-sensitive PPAP2B.

PPAP2B suppression abrogated athero-protection of unidirectional flow, shown by elevation of inflammatory genes. Inhibition of lysophosphatidic acid receptor 1 restored the flow-dependent, anti-inflammatory phenotype in PPAP2B-deficient cells. Moreover, PPAP2B inhibition resulted in myosin-light-chain phosphorylation and intercellular gaps, which were abolished by inhibition of lysophosphatidic acid receptors 1 and 2. Expression-quantitative-trait-locus-mapping demonstrated PPAP2B CAD risk allele is not linked to PPAP2B expression in various human tissues but significantly associated with reduced PPAP2B in HAEC.

Conclusions:
Athero-relevant flows dynamically modulate endothelial PPAP2B expression through miR-92a regulation. Mechano-sensitive PPAP2B plays an indispensable role in mediating cell alignment, promoting anti-inflammatory phenotype and maintaining vascular integrity of endothelial monolayer under athero-protective flow.
Abstract 373: Transgenic Overexpression of Alanine-glyoxylate Aminotransferase 2 Lowers Tissue Levels of Asymmetric Dimethylarginine and Improves Endothelial Function in Mouse Aortas
Authors: Rodionov Roman N , Burdin Dmitrii V , Todorov Vladimir , Brilloff Silke , Jarzebska Natalia , Martens-Lobenhoffer Jens , Hofmann Anja , Morawietz Henning , Demyanov Anton V , Maas Renke , Bode-Böger Stefanie M , Hugo Christian P , Hohenstein Bernd , Weiss Norbert
Published: July 2021

Introduction:
Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide synthase, which has been proposed to play a direct role in the pathogenesis of cardiovascular disease. There are two enzymatic pathways for degradation of ADMA: hydrolysis to citrulline by dimethylarginine dimethylaminohydrolase (DDAH) and transamination by alanine-glyoxylate aminotransferase 2 (AGXT2) with formation of asymmetric dimethylguanidino valeric acid (ADGV). The first pathway is well characterized, whereas the physiological role of AGXT2 is still poorly understood. The goal of our study was to test the hypothesis that transgenic overexpression of AGXT2 would lead to lowering of systemic levels of ADMA and improved vasomotor function.

Methods and results:
We generated transgenic mice (TG) with ubiquitous overexpression of AGXT2 under control of the chicken beta actin (CAG) promoter. Ubiquitous overexpression of the transgene was confirmed by qPCR and Western Blot. TG animals had normal weight and no observed developmental abnormalities. Biochemical data were generated using HPLC-MS/MS. ADMA plasma levels in AGXT2 TG animals were decreased by 15% (p<0.05), whereas ADGV levels were 6 times higher in TG animals in comparison with wild-types (p<0.001). Lung and heart of TG animals exhibited 2 times lower tissue ADMA content in comparison with controls (p<0.05). Isolated aortic rings were used to estimate endothelium-dependent and -independent relaxation in response to acetylcholine (Ach) and sodium nitroprusside (SNP), respectively. Aortas from AGXT2 TG mice demonstrated an increase in maximal response to ACh (p<0.05). There was a similar relaxation in response to SNP in both groups.

Conclusions:
Our findings show that upregulation of AGXT2 results in lower ADMA levels and improved endothelial-dependent relaxation in vivo. AGXT2 thereby may be a therapeutic target for long-term reduction of systemic ADMA levels and improvement of vascular function in vivo. This is especially important, because all the efforts to develop ADMA-lowering interventions by means of upregulation of DDAH have not been successful so far. Our data suggest that AGXT2 might be a promising drug target for cardiovascular pathologies associated with elevated ADMA levels.
Abstract 382: Lipin-1 Links Pro-inflammatory Responses and Foam Cell Formation by Oxidized-Low Density Lipoprotein-Elicited Macrophages
Authors: Navratil Aaron R , Vozenilek Aimee E , Cardelli James A , Green Jonette M , Orr A W , Woolard Matthew D
Published: July 2021

Atherosclerosis is a chronic inflammatory disease of large and medium-sized arteries and one of the underlying causes of cardiovascular disease (CVD). Macrophages participate decisively in the development and promotion of atherosclerosis. Macrophages infiltrate the arterial intima to ingest modified low density lipoproteins (e.g. oxLDLs) via scavenger receptors. The scavenging of oxLDLs results in foam cell formation due to enhanced lipid droplet biogenesis. These foam cells eventually release pro-inflammatory cytokines that promote atherosclerosis. However, it is currently unknown whether there is a link between lipid droplet biogenesis and pro-inflammatory cytokine production in macrophages that scavenge oxLDL. Lipin-1, a phosphatidate phosphohydrolase enzyme, partially contributes to macrophage pro-inflammatory cytokine production following stimulation with bacteria. Lipin-1 is also required for lipid droplet biogenesis in macrophages. Finally, we observed lipin-1 protein within macrophages from human atherosclerotic plaques. Thus, we hypothesized that lipid droplet biogenesis, via lipin-1 activity, directly contributes to foam cell pro-inflammatory cytokine production. To test this hypothesis we compared lipid droplet biogenesis and pro-inflammatory cytokine responses of oxLDL-stimulated wild type and lipin-1-depleted macrophages. Depletion of lipin-1 inhibited oxLDL-induced foam cell generation by reducing lipid droplet number, area, and staining intensity. There were no differences in scavenger receptor expression or uptake of oxLDL between wild type and lipin-1-depleted cells. In addition, depletion of lipin-1 also ablated oxLDL-elicited production of the pro-atherogenic cytokines tumor necrosis factor-α and interleukin-6. These findings demonstrate a critical role for lipin-1 in the regulation of macrophage inflammatory responses to oxLDL. Furthermore, these data begin to link foam cell formation, via lipid droplet biogenesis, and pro-inflammatory cytokine production within oxLDL stimulated macrophages. Thus, our studies suggest that lipid droplet biogenesis may be an ideal therapeutic target to inhibit inflammation associated with atherosclerosis to treat CVD.
Abstract 393: Reticulon-4B Protects Against Endoplasmic Reticulum Stress Induced Platelet Apoptosis and Hyperactivation in Diabetes
Authors: Wang Yan , Tang Wai Ho , Zhang Xinbo , Du Jing , Hwa John , Yu Jun
Published: July 2021

Background:
Hyperglycemia triggered endoplasmic reticulum (ER) stress is one of the major causes for platelet hyperactivation and apoptosis in diabetes mellitus (DM). Reticulon-4B (aka Nogo-B) mainly localizes to the ER, and has been shown to influence the ER morphology, ER-Golgi trafficking, apoptotic balance, vesicle formation and protein trafficking in cells. The present study is aimed to investigate the role of Nogo-B on platelet function in DM.

Methods and Results:
Nogo-B is highly expressed in platelets from healthy individual. Platelets from DM patients and diabetic mice have decreased Nogo-B level. Using Streptozotocin (STZ) induced diabetic mouse model, we show that loss of Nogo (Nogo-/- mice) decreased platelet number, increased mean platelet volume and prolonged bleeding time compared to wild-type (WT) mice. Platelets from Nogo-/- mice were hyperactive with higher JONA and P-selectin surface expression compared to WT mice. Loss of Nogo increased thrombin and collagen induced platelet aggregation. Furthermore, platelets from diabetic Nogo-/- mice show elevated reactive oxygen species (ROS) production, decreased mitochondria membrane potential and increased apoptosis, which can be rescued by antioxidant N-acetyl-L-cysteine. Mechanistically, we show Nogo-B prevented sequestration of antiapoptotic proteins Bcl-xL and Bcl-2 induced by hyperglycemia, subsequently protected against platelet mitochondrial damage, ROS production, caspase-3 activation and apoptosis.

Conclusion:
These findings demonstrate that Nogo-B protects against ER stress induced platelet apoptosis and hyperactivation in DM by regulating Bcl-xL and Bcl-2 sequestration and mitochondrial damage. This novel pathway may provide therapeutic targets for thrombotic complications in diabetes mellitus.
Abstract 404: The Role of 20-HETE in the Regulation of Endothelial Progenitor Cell Stemness
Authors: Joseph Gregory , Chen Li , Falck John R , Schwartzman Michal L , Guo Austin M
Published: July 2021

Objective:
Endothelial progenitor cells (EPC) are known to contribute to neovascularization by producing various angiogenic cytokines, such as VEGF, at target sites. Factors that are capable of maintaining EPC stemness can in turn promote EPC self-renewal, thereby increasing their angiogenic functions. We recently identified the CYP4A-derived 20-HETE regulates some of the EPC pro-angiogenic properties (e.g. proliferation, migration)
in vitro
. In the present study, we further investigated the role of 20-HETE in the regulation of EPC stemness that are associated with their angiogenic functions.

Methods:
Human umbilical cord blood-derived EPC were isolated by magnetic cell sorting and cultured in an EPC enrichment media. LC/MS/MS was performed in cells harvested at various times during the EPC-EC differentiation process for 20-HETE production. Next, real-time PCR was used to assess the effects of 20-HETE (1 nM) on the expression of Oct4, Sox2, and NANOG, genes commonly associated with cell stemness. Flow cytometry was performed to quantify the population of progenitor
vs
differentiated EPC in the presence and absence of a 20-HETE synthesis inhibitor (DDMS; 10 μM) or a 20-HETE competitive antagonist (6, 15-20-HEDGE; 10 nM) in EPC cultures. Lastly, the effects of global increase in 20-HETE induction
in vivo
on bone marrow (BM)-derived EPC stemness were also examined using flow cytometry.

Results:
We found that differentiation of EPC to EC was associated with a time-dependent decrease in 20-HETE production. Exogenous 20-HETE significantly increased the expression of Oct4, Sox2, and NANOG by 1.5-, 2-, and 8.2-fold, respectively. DDMS and 6, 15-20-HEDGE decreased the number of progenitor cell population compared to vehicle-treated controls. In contrast, addition of 20-HETE increased the number of cell population expressing progenitor cell markers. Finally, BM-derived EPC from high 20-HETE production mice CYP4a12tg contained 50% more progenitor cells than EPC derived from control animals.

Conclusion:
These results implicate 20-HETE as a novel regulator of EPC stemness and an important player in the regulation of angiogenic responses that require the contribution of EPC.
Abstract 479: Orotic Acid Induces Hypertension Associated With Impaired Endothelial Nitric Oxide Synthesis
Authors: Choi You-Jin , Lee Byung-Hoon
Published: July 2021

Orotic acid (OA) is an intermediate of pyrimidine nucleotide biosynthesis. Hereditary deficiencies in some enzymes associated with pyrimidine synthesis or the urea cycle induce OA accumulation, resulting in orotic aciduria. A link between patients with orotic aciduria and hypertension has been reported; however, the molecular mechanisms remain elusive. In this study, to elucidate the role of OA in vascular insulin resistance, we investigated whether OA induced endothelial dysfunction and hypertension. OA inhibited insulin- or metformin-stimulated nitric oxide (NO) production and endothelial NO synthase (eNOS) phosphorylation in human umbilical vein endothelial cells (HUVEC). A decreased insulin response by OA was mediated by impairment of the insulin-stimulated phosphoinositide 3-kinase (PI3K)-protein kinase B (PKB/Akt) signaling pathway in cells overexpressing the p110-PI3K catalytic subunit. Impaired effects of metformin on eNOS phosphorylation and NO production were reversed in cells transfected with constitutively active AMP-activated protein kinase. Moreover, experimental induction of orotic aciduria in rats caused insulin resistance, measured as a 125% increase in the homeostasis model assessment, and hypertension, measured as a 25% increase in systolic blood pressure. OA increased the plasma concentration of endothelin-1 by 201% and significantly inhibited insulin- or metformin-induced vasodilation. A compromised insulin or metformin response on the Akt/eNOS and AMPK/eNOS pathway was observed in aortic rings of OA-fed rats. Taken together, we showed that OA induces endothelial dysfunction by contributing to vascular and systemic insulin resistance that affects insulin- or metformin-induced NO production, leading to the development of hypertension.
Abstract 497: 5-Methoxytryptophan Protects Against Vascular Remodeling
Authors: Ho Yen-Chun , Wu Meng-Ling , Su Chen-Hsuan , Kuo Cheng-Chin , Wu Kenneth K , Yet Shaw-Fang
Published: July 2021

Vascular smooth muscle cells (VSMCs) in the blood vessel wall exhibit a differentiated phenotype; their main function is contraction and to regulate vascular tone. In response to injury, VSMCs undergo a phenotypic transition whereby they proliferate and migrate from the medial layer into the intima, contributing to lesion formation and atherosclerosis. 5-methoxytryptophan (5-MTP), a recently identified novel tryptophan metabolite, has been shown to inhibit cancer cell growth, migration, and cancer metastasis. We hypothesized that 5-MTP might play an analogous role in vascular disease as in tumorigenesis. To test our hypothesis, we subjected 12 weeks old C57BL/6 mice to a carotid artery cessation of blood flow model to induce neointima formation. Following surgery, mice were treated with vehicle (PBS) or 100 mg/kg of 5-MTP by intraperitoneal injection 3 times a week. Four weeks later, carotid arteries were harvested for histological analysis. H&E and elastin staining revealed robust neointima in ligated carotids of vehicle-treated mice. In contrast, 5-MTP significantly attenuated neointima formation. Given that proinflammatory cytokine interleukin 1 beta (IL-1β) is increased in the injured vessel wall, we examined whether IL-1β affected VSMC phenotypic modulation. Indeed, IL-1β downregulated VSMC marker SM α-actin expression and increased VSMC proliferation and migration. Importantly, in VSMCs, 5-MTP attenuated IL-1β-mediated SM α-actin downregulation, proliferation, and migration, suggesting a potential role of 5-MTP in controlling VSMC phenotypic modulation. Furthermore, 5-MTP inhibited IL-1β-mediated p38 MAPK activation. Inhibiting p38 MAPK activation by SB203580 dose-dependently decreased IL-1β-induced VSMC proliferation. Taken together, our results suggest an important role of 5-MTP in vascular disease.