bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 527: The BMP Inhibitor DMH1 Inhibits Aortic Smooth Muscle Cell Calcification
Authors: Lin Tonghui , Wang Xue-lin , Guzman Raul
Published: July 2021

Arterial calcification is associated with increased morbidity and mortality in patients with lower extremity occlusive disease. In calcifying arteries, vascular smooth muscle cells (SMC) undergo phenotypic transformation to a more bone-like cell. It is thought that Bone Morphogenetic Proteins (BMPs) may regulate this process. We sought to evaluate the effects of the novel, synthetic, non-peptide, BMP inhibitor DMH1 on SMC calcification.

Methods:
Confluent human aortic SMCs were incubated in calcification medium containing 3 mM Phosphate (Pi). Cells in calcification medium were treated with increasing concentrations of DMH1. After 7 days, cells were incubated in 0.6N HCl overnight and the amount of soluble calcium was measured in the media. Calcium levels were normalized to protein content as measured using the BCA assay. Total RNA from SMCs was isolated with RNeasy mini kit and qRT-PCR was performed with respective gene-specific primers. Rat aorta rings were induced to calcify by stimulation with phosphate in DMEM with or without DMH1 for 7days. Calcium was measured as described above. Changes in BMP signaling proteins p-smad1/5/8 and total smad1/5/8 were assessed by western blot at 24 hours.

Findings:
Cells grown in calcification medium had increased amounts of calcification compared with controls; however, this was significantly reduced by addition of DMH1 in a dose dependent manner. Similar findings were seen in aortic rings. Interestingly, DMH1 increased the expression of the two bone-related proteins alkaline phosphatase and RANK ligand while decreasing the expression levels of the SMC-specific genes α-SM-actin and SM22-α. Western blot analysis revealed decreased p-smad1/5/8 levels in DMH1-treated cells.

Conclusions:
The synthetic BMP inhibitor DMH1 inhibits Pi-induced calcification of human vascular SMCs and rat aorta rings. BMP-inhibiting compounds may have clinical utility in inhibiting arterial calcification and reducing amputation in our patients with critical limb ischemia.
Abstract 529: Cbfa1 (Runx2) is a Mediator of Aortic Stiffness and Hypertension in a Murine Model of Diabetes Mellitus Type 2
Authors: Raaz Uwe , Schellinger Isabel N , Adam Matti , Nakagami Futoshi , Deng Alicia , Emrich Fabian C , Jagger Ann , Kayama Yosuke , Findley Clarence , Spin Joshua M , Tsao Philip S
Published: July 2021

Accelerated arterial stiffening is a complication of diabetes mellitus and associated with the development of hypertension. Arterial stiffening results from extensive extracellular matrix remodeling (elastin breakdown, collagen accumulation). The osteogenic transcription factor Cbfa1 (Runx2) has been identified as a mediator of aortic calcification and regulator of matrix protein expression. However, its impact on aortic stiffness is unclear.

This study was designed to elucidate the temporal relation between aortic stiffening and the development of arterial hypertension in a murine model of diabetes mellitus type 2. Moreover we aimed to investigate the role of Cbfa1 as potential mediator diabetic aortic stiffening.

Serial ex vivo mechanical testing of the thoracic aorta and volume-pressure recording (VPR) based tail-cuff blood pressure measurements revealed that aortic stiffening precedes blood (pulse) pressure elevations in diabetic db/db mice. Vascular stiffening was accompanied by increased medial collagen deposition Picrosirius Red staining). qRT-PCR and immunohistochemistry revealed enhanced expression of Cbfa1 and target genes (Col1a1, Col1a2, FN1, Spp1) in db/db mice compared to controls. Moreover, overexpression of Cbfa1 in vascular smooth muscle (Cbfa1-smTg mice) results in increased medial collagen deposition, aortic stiffness and augmented pulse pressure. Interestingly, Cbfa1-smTg mice did not exhibit enhanced vascular calcification (by von Kossa and Alizarin Red staining).

In conclusion we demonstrated that aortic stiffening precedes the onset of hypertension in db/db mice and identified Cbfa1 as mediator of aortic stiffening - presumably via pro-fibrotic mechanisms.
Abstract 541: Specific Deletion of LDL Receptor--Related Protein on Macrophages Skews Cytokine Production by Invariant Natural Killer T Cells
Authors: Covarrubias Roman , Major Amy S
Published: July 2021

Invariant Natural Killer T (iNKT) cells are specialized lymphocytes that when activated can regulate chronic inflammatory conditions and atherosclerotic processes. The activation of iNKT cells occurs when glycolipid antigens bind the MHC class-I like molecule CD1d present on antigen presenting cells (APCs). The pathways by which glycolipid antigens target CD1d for presentation and activation of iNKT cells remain unclear, yet the expression of surface receptors associated with lipid homeostasis, such as the LDL receptor (LDLr), have been implicated in the modulation of iNKT cell activation. The LDLr has been shown to modulate this process by binding apoE-containing lipoproteins, which can carry antigenic glycolipids for iNKT cell activation. The LDL receptor-related protein (LRP), a transmembrane receptor from the LDL receptor family of proteins, shares structural homology with LDLr and can bind a number of ligands including apoE-containing lipoproteins. We hypothesized that LRP can play an active role in glycolipid antigen presentation and subsequent activation of iNKT cells. Here, we demonstrate that LRP is preferentially expressed at high levels on F4/80
+
macrophages, when compared to other APCs. We also show that a specialized subset of macrophages expressing CD169, known for their ability to present glycolipid antigen to iNKT cells, have increased levels of LRP when compared to CD169
-
macrophages. Using mice with a targeted deletion of LRP in macrophages, we observed decreased activation of iNKT cells
in vitro
(24, 48 hours) and normal IFN-gamma but blunted IL-4 response
in vivo.
Further flow cytometric analysis showed normal surface expression of CD1d in LRP-cKO macrophages as well as normal uptake of fluorescently labeled glycolipid
in vitro
. Additionally, analysis of the iNKT cell compartment in LRP-cKO mice revealed intact numbers and percentages of iNKT cells and no homeostatic disruption as evidenced by absence of programmed death-1 and LY-49. Collectively, these data suggest that macrophage LRP contributes to early iNKT cell activation by enhancing early IL-4 responses.
Abstract 544: Nrp-1+Foxp3-CD4 T Cells Are a Novel Subset of T Lymphocytes that Are Induced in Aorta During Development of Atherosclerosis
Authors: Gaddis Dalia E , Miller Yury , Sorci-Thomas Mary , Hedrick Catherine C
Published: July 2021

Neuropilin 1 (Nrp-1) is a type I transmembrane protein that plays an important role in axonal guidance and neuronal development as well as vascular sprouting and angiogenesis through the interaction with Semaphorin-3A and vascular endothelial growth factor (VEGF), respectively. Recently, Nrp-1 was found on a subset of thymus-derived regulatory T cells (Treg) that migrated into VEGF producing tumors. Moreover, Nrp-1+ CD4 T cells were protective in a murine experimental model of autoimmune encephalomyelitis, suggesting that these cells may have a participating role in other inflammatory diseases. Since atherosclerosis is a chronic inflammatory disease where T cells, macrophages, and other immune cells orchestrate disease progression, we hypothesized that Nrp-1+ CD4 T cells are induced in the aorta during atherosclerosis development and play a role during disease progression. We fed ApoE-/- mice a western diet for 15 weeks and found a 2-fold increase in Nrp-1+Foxp3- CD4 T cells in the spleens, peri-aortic lymph nodes and aortas of western diet fed mice compared to chow-fed mice. In addition, these Nrp-1+Foxp3- CD4 T cells expressed higher levels of the memory marker CD44 and produced more IFNγ when compared to Nrp-1- CD4 T cells, suggesting that these cells are more activated. Treatment of CD4 T cells with oxidized LDL (OxLDL), but not minimally modified LDL (mmLDL), oxidized cholesterol ester (OxCE) or oxidized phospholipids (POVPC), caused upregulation of Nrp-1 on CD4 T cells. Using a transwell migration assay, we found that Nrp1+ CD4 T cells had a 2-fold higher migration index towards VEGF165, a splice variant of VEGF, than did Nrp1- CD4 T cells; thus this migration was dependent on Nrp-1 expression. Similarly, CD4 T cells from ApoE-/- mice fed a western diet, which expressed more Nrp-1, had a higher migration index towards VEGF165 compared to cells from chow controls. In conclusion, we have identified a novel subset of Nrp-1+CD4+ T lymphocytes that is increased during atherosclerosis development, and is induced by OxLDL. Our data suggest that Nrp-1 may play a role in the migration of CD4 T cells to the aorta during atherosclerosis development.
Abstract 548: TLT-1 as a Possible Modulator of Systemic Lupus Erythematosus
Authors: Morales-Ortiz Jessica , Yerania Rodríguez-Navedo Yerania , Vilá-Rivera Karina , Nieves-Plaza Mariely , Ricaute Marta , Vilá Luis M , Washington Anthony V
Published: July 2021

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by immune system hyperactivity and autoantibody production that can affect almost any organ system. TREM like transcript-1 (TLT-1) is a membrane receptor found in platelets α-granules with activating properties. We have demonstrated that activated platelets release a soluble isoform of TLT-1 (sTLT-1) which is not found in the plasma of healthy individuals. Moreover, evaluation of patients suffering from inflammatory diseases, such as sepsis, reveals a significant increase of plasma sTLT-1 levels. It is not known if TLT-1 plays a role in auto immune diseases such as Lupus. In the present study we evaluate the potential that TLT-1 may play a role in the etiology of SLE. We assessed the plasma of 46 SLE patients and compared to 28 healthy controls by ELISA. Our results showed that SLE patients had a significant decrease in plasma sTLT-1 levels compared to healthy controls (9.0 [7.2] vs. 18.6 [22.3] ng/ml, p=0.008). A negative correlation was observed between TLT-1 levels and LupusPRO [lupus symptoms (r = -0.388, p = 0.055). Based on this data;
we hypothesize that SLE patients produce autoantibodies against TLT-1 in serum which might correlate with clinical aspects of the disease
. To address this question; TLT-1 antibodies levels in serum were measured by ELISA. Our data suggests that a sub-population of SLE patients have TLT-1 autoantibodies. This data was further confirmed by confocal microscopy. Using human embryonal kidney cells (HEKt) stably transfected with TLT-1, we evaluated the serum of SLE patients for the presence of TLT-1 autoantibodies using immunofluorescence. Our data confirms that a sub-population of SLE patients with active disease is expressing antibodies against TLT-1. These findings suggest a possible role for TLT-1 during the pathogenesis of SLE and targets new treatment possibilities for lupus patients.
Abstract 551: In a South Asian Population, Urbanization is an Independent Predictor of Arterial Stiffness in Men
Authors: Thanikachalam Mohan , Lane Kevin , Sunderarajan Jahnavi , Corlin Laura , Harivanzan Vijaykumar , Brugge Doug , Muralidharan T R , Thanikachalam Sadagopan
Published: July 2021

Background:
Rapid urbanization is driving economies of South Asian countries. Here we use satellite based land cover data and distance to urban center (UC) to measure of the impact of urban environment on arterial stiffness (AS) in a population based study in South India.

Methods:
In a cross-sectional analysis, after exclusion of people with previous history of diabetes and hypertension, 6746 subjects (mean age 42 years; 54% women) spread over 78 kms from the UC constituted the study sample. MODIS satellite derived land cover data at a 1 km x 1 km resolution was obtained and joined to each participant's geolocated residential position in ArcGIS to assign urban and rural designations. The study included carotid-femoral pulse wave velocity (PWV) measurement using a high-fidelity applanation tonometry, blood pressure (BP), anthropometric, psychosocial, high sensitive C-reactive protein (HsCRP) and other biomarkers assessments.

Results:
Based on land cover analysis, participants in urban locations had a mean (SD) PWV (m/s) of 7.74 (1.65) compared to 7.6 (1.62) in rural locations (p= 0.002) [Fig 1], while there was no significant difference in HsCRP levels. In multiple regression analyses adjusting for age, smoking, BMI, BP, blood glucose, LDL, socioeconomic, anxiety and stress levels, distance from UC was independently associated with PWV in men (β = -0.007, p <0.001), but not in women. Standardized effect-estimates in the multi-linear regression model indicated that distance from UC had the third largest effect on PWV after age and BP. After multivariable adjustments, the largest effect of distance from UC on PWV was on non-smoking men age 46-75 years. Residing every 1 km further away from the UC corresponded with a -0.012 m/s (95%CI: -0.020, -0.003) decrease in PWV.

Conclusions:
Urbanization is an independent predictor of AS in men, more so in non-smoking older men. Further research will elucidate components in the urban environment that may be contributing to higher AS.
Abstract 568: Biomarkers of Hemostasis and Thrombosis in Hemophilia A and B Patients
Authors: Chen Zhu , Wu Weizhen , Ge Lan , Ichetovkin Marina , Desai Kunal , Wang Weixun , Xu Yiming , Ogletree Martin L , Seiffert Dietmar A
Published: July 2021

The defective hemostatic process in hemophiliacs overlaps with the underlying physiology contributing to bleeding risk in anticoagulated patients. Hemophiliacs may also develop compensatory changes in fibrinolysis, platelet reactivity, vascular inflammation, and remodeling secondarily. We therefore explored markers for these processes in hemophilia A and B as part of our larger effort to develop translational biomarkers to guide antithrombotic therapy and minimize bleeding risk.

Plasmas from hemophilia A patients (11 severe; 1 moderate) and hemophilia B patients (3 severe; 3 mild or moderate), sampled after a treatment washout period, and 7 normal subjects were procured. Markers/assays pursued include: coagulation (F1+2, FPA, TAT, D-dimer), fibrinolysis (PAP, PAI-1, tPA), platelet activation (soluble P-selectin (sP-sel)), vascular inflammation (MCP-1), angiogenesis (VEGF), PT, aPTT, and thrombin generation assay (TGA). Consistent with aPTT prolongation and changes in TGA, both hemophilia groups displayed numerically lower than normal levels of all 4 coagulation markers, with hemophilia A reaching statistical significance for F1+2 and TAT. Hemophilia A also exhibited a significant decrease in PAP and a numerical increase in PAI-1 compared to normal. VEGF and MCP-1 exhibited no appreciable difference. In the combined analysis for hemophilia A and B (“A+B”), PAP displayed a strong positive correlation with F1+2 and a strong negative correlation with PAI-1; A+B exhibited a statistically significant reduction in F1+2, FPA, and TAT, and a non-significant increase in sP-sel and non-significant decrease in PAP, compared to normal.

In conclusion, hemophilia A and B plasma samples had significantly reduced markers of in vivo thrombin generation, thrombin activity, and coagulation, and trends evidencing increased platelet activation and decreased fibrinolysis, possibly as compensation. Lack of signal in vascular inflammation or angiogenesis could be due to the (near optimal) hemostatic therapies. Our results illustrate the potential value of biomarkers of in vivo coagulation, fibrinolysis, and platelet activation in translational research on antithrombotic therapies for both their efficacy and bleeding risk.
Abstract 585: Endotoxin Contamination of Apolipoprotein A1: Effect on Macrophage Proliferation---a Cautionary Tale
Authors: Jin Xueting , Xu Qing , Champion Keith , Kruth Howard S
Published: July 2021

Incubation of human macrophages with ApoA1 (purified from human plasma) for 1-2 days resulted in decreased cell density that was independent of cell toxicity. Although ApoA1 treatment caused macrophages to become more rounded and clustered, with some macrophages detaching, this did not account for the decrease in cell density. Thus, we considered that ApoA1 decreased macrophage cell proliferation. Using a BrdU incorporation cell proliferation assay, we demonstrated that ApoA1 (20 ug/ml) resulted in a >90% inhibition in macrophage proliferation. Using a recombinant version of ApoA1, we found that the results were dependent on the expression system: ApoA1 expressed in human cells did not inhibit proliferation but ApoA1 expressed in E. coli did. Human plasma-derived ApoA2 did not inhibit proliferation. All apolipoproteins were obtained commercially.

We considered the possibility that endotoxin contamination of the apolipoproteins contributed to the differential inhibition of macrophage cell proliferation. Endotoxin alone very potently inhibited macrophage proliferation (0.1 ng/ml inhibited macrophage proliferation >90%). Measurement of endotoxin levels in the apolipoprotein products, including an analysis of free versus protein-bound endotoxin, suggested endotoxin mediated inhibition of macrophage proliferation. While human cell-derived recombinant ApoA1 and human plasma-derived ApoA1 showed similar levels of total endotoxin contamination (levels that were sufficient to inhibit macrophage proliferation), the levels of free endotoxin in the human cell-derived recombinant ApoA1 were more than 1000-fold less than levels in the human plasma-derived ApoA1. This finding helps explain why the recombinant ApoA1 did not inhibit macrophage proliferation, but the plasma-derived ApoA1 did.

Conclusion:
Our findings show that apparent apolipoprotein-mediated cell effects (or effects of any other biological products) can be significantly influenced by endotoxin contamination, especially when these products are tested on highly endotoxin-sensitive cells, such as macrophages. Lastly, endotoxin potently inhibits macrophage proliferation, which may be a significant pathogenic mechanism by which bacteria escape the immune system.
Abstract 592: Nutrigenomic Screening of Functional Foods Regulating Gluconeogenesis: A Zebrafish Pilot Study
Authors: Bai Ji Dong , Wang Youdong , Wen Xiao-Yan
Published: July 2021

Introduction/rationale:
Current anti-diabetic drug treatments have a variety of adverse side effects. Identifying functional foods with anti-diabetic properties may be the key to preventing and managing T2DM while minimizing side effects. Studies found that Pck-1 gene is often down-regulated by anti-diabetic drugs, as its enzyme is responsible for catalyzing the rate-limiting step of gluconeogenesis. In our study, we screened for functional foods that can down-regulate the expression of the Pck-1 gene. The newly emerged zebrafish (Danio rerio) is aptly suited for in vivo nutrigenomic screening due to its large clutch size and conservation of molecular pathways with humans, including those involved in glucose regulation.

Methods:
We exposed zebrafish larve (Tg(Pck-1:luc), a luminescent reporter line for Pck-1), at 4 days post fertilization (dpf) to extracts from forty functional fruits and vegetables. The level of Pck-1 expression was quantified by luminescence at 6 dpf. Extracts which resulted in a lower reading of luminescence were interpreted as functional foods that could successfully down-regulate Pck-1 expression, and therefore may be potential therapeutic for T2DM. We further validated our results using a fluorescent reporter line for Pck-1 (Pck-1:eGFP).

Results:
We completed toxicity assays on the functional food extracts to determine the optimal concentrations and conditions for nutrigenomic screening. Preliminary results show that bamboo and cherry extracts significantly reduced Pck-1 gene expression by two fold (p<0.05). Other functional food extracts, such as grape, cucumber, cabbage, spinach, flat beans, and lemon also reduced Pck-1 gene expression. Interestingly, garlic extracts increased Pck-1 gene expression.

Conclusion and Discussion:
We identified two functional food extracts that successfully reduced Pck-1 expression in zebrafish models. Increasing the intake of these two functional foods may regulate blood glucose levels in T2DM patients. Further research elucidating the active ingredient in these foods is required. The Pck-1 zebrafish model can be employed for further nutrigenomic screening and/or for drug discovery purposes.
Abstract 597: Nobiletin Reduces Intestinal Lipoprotein Production in LDL Receptor-Null Mice With Diet-Induced Insulin Resistance
Authors: Chhoker Sanjiv S , Sutherland Brian G , Telford Dawn E , Huff Murray W
Published: July 2021

Insulin resistant subjects display excessive postprandial lipemia (PPL), defined by overproduction of intestinal triglyceride (TG)-rich, apoB48-containing chylomicrons (CM), which contribute to atherogenesis. However, the regulation of intestinal lipoprotein secretion by insulin and the mechanisms underlying elevated CM production in insulin resistant states remain poorly understood. In mice, the citrus flavonoid nobiletin prevents diet-induced obesity, dyslipidemia, insulin resistance, hepatic steatosis and VLDL overproduction. In the present study, we sought to characterize the aberrant PPL response in mice with diet-induced insulin resistance and hypothesized that nobiletin treatment would correct this abnormal response by normalizing intestinal insulin signaling and TG metabolism. Ldlr-/- mice were fed a high-fat, cholesterol-containing (HFHC) diet (42% fat, 0.2% cholesterol), a HFHC diet plus 0.3% w/w nobiletin or chow for 8 weeks (n=8 per group). Compared to chow, HFHC-fed mice displayed elevated TG (3-fold) within intestinal tissue following a 6 hour fast, which was completely normalized by nobiletin. Fasting-refeeding studies revealed impaired FoxO1 phosphorylation, increased mTOR phosphorylation and elevated Srebp1c mRNA in the jejunum of HFHC-fed mice, indicating intestinal insulin resistance. These parameters were normalized by nobiletin. Measurement of plasma TG following an oral gavage of 150ul olive oil revealed a 3-fold elevation in the total TG area under the curve, which was prevented by nobiletin. Intestinal CM production was determined by injection of mice with poloxamer-407 to inhibit plasma TG-rich lipoprotein lipolysis followed by a gavage containing olive oil and 3H-triolein. Although plasma TG was higher at baseline in HFHC-fed mice, no difference in secretion rates of TG mass or TG radioactivity was observed compared to nobiletin-treated or chow-fed mice. However, secretion of apoB48 was elevated by 20% in HFHC-fed mice, whereas nobiletin decreased apoB48 secretion to levels observed in chow-fed mice. These results indicate that nobiletin prevents intestinal TG accumulation and apoB48 overproduction induced by a HFHC diet, in part, through correction of intestinal insulin resistance.
Abstract 615: Rigorous Exercise and Quercetin Intake Modulate Adiponectin and Reduce Body Weight in Normal C57BL6 Mice
Authors: Mahini Halleh , Ainsworth Gregory , Garelnabi Mahdi
Published: July 2021

Introduction:
Adiponectin has been suggested to play an important role in the modulation of glucose and lipid metabolism in in both humans and animals. We previously reported that intake of quercetin during exercise decreases body weight in LDL -/- C57BL6 mice. This finding has encouraged us to expand the investigation by increasing the exercise duration, intensity and also by testing these effects in normal C57BL mice

Aims and Objectives:
In the current study we proposed that quercetin will have profound effects on the modulators of adipose tissue if combined with exercise, we hypothesized that this may be the major mechanism that contribute to the body weight reduction seeing in our previous findings.

Study Design:
40 mice were divided into four groups (10 each). These groups are as follows: Control mice, left untreated; control quercetin group, orally supplied with 100 μg/day of quercetin without exercising; exercise group without quercetin, and exercise group with quercetin supplements. The exercise groups were run on a treadmill for 30 minutes, 20-30m/m/ 5 days/week for two month. All animals were on normal mouse chow. At the end of the two month treatment, tissues gene expressions for genes associated with obesity, oxidative stress, inflammation and lipoproteins were analyzed. We also measured plasma markers such as paraoxonase-1 enzyme activity and adiponectin

Results:
Data from the current study confirmed our previous findings that the combination of the intake of quercetin and exercise reduces body weight. This study demonstrated a significant (P<0.0009) drop in changes of body weight (43.5%) compared to control. Mice on quercetin intake without exercise had 29% drops in body weight. Conversely in the current study all the three groups illustrated significant changes in the plasma adiponectin levels compared to the control. Plasma diponectin levels in mice on exercise regimen with or without quercetin intake, or only quercetin intake have significantly (P<0.007) decreased compared to the controls.

Conclusion:
The combination of exercise and quercetin or separately modulate plasma adiponectin levels. An adiponectin dependent mechanism may be responsible for the reduction in body weight observed in this study with the combination of exercise.
Abstract 641: Interleukin-15 Plays a Role in Atherosclerosis Through Effects on Multiple Inflammatory Cells in ApoE-/- Mice
Authors: Dadoo Omid , Ashkar Ali , Richards Carl , Trigatti Bernardo
Published: July 2021

Objective:
Interleukin (IL)-15 is a pro-inflammatory cytokine which is essential for the survival and maturation of natural killer (NK) and CD8+ T cells and has recently been reported to directly activate macrophages. In the present study, we used IL-15 overexpressing and IL-15 knockout mice to determine its effect on atherosclerosis in apolipoprotein E knockout (apoE-/-) mice.

Approach and Results:
Deficiency of IL-15 reduced atherosclerosis while overexpression of IL-15 increased atherosclerosis despite opposite effects on plasma lipoprotein cholesterol levels. Genetic ablation of IL-15 in apoE-/- mice resulted in a lack of NK cells and reduced populations of monocytes and CD8+ T cells, whereas overexpression of IL-15 had the opposite effects. CD8+ T cells were detected in atherosclerotic plaques from apoE-/- mice, were increased in plaques from il-15TgapoE-/- mice and were virtually absent from plaques from il15-/- apoE-/- mice. To test the role of NK, NKT and activated CD8+ T cells in atherosclerosis, NK1.1+ cells were immuno-depleted from apoE-/- mice. Atherosclerosis was reduced but to a lesser extent than in age matched il15-/- apoE-/- mice. Plaques from these mice, however, were almost devoid of CD8+ T cells. ApoE-/- mice that were heterozygous for IL-15 (il15+/-apoE-/-) exhibited normal levels of NK, NKT, CD8T cells and monocytes however atherosclerosis was reduced by 40-50 % compared to control apoE-/- mice. This suggested that IL-15 also affects atherosclerosis through NK/NKT and CD8+ T cell independent pathways. Treatment of macrophages with recombinant IL-15 in vitro induced inflammatory cytokines and foam cell formation. Furthermore, reduced levels of MCP-1 and CD11b immune-reactivity were detected in plaques from il15-/- apoE-/- compared to control apoE-/- mice.

Conclusions:
IL-15 plays a significant role in promoting atherosclerosis through pathways affecting multiple inflammatory cells including the survival/recruitment of NK, NKT and CD8+ T cells, the modulation of monocyte levels, and direct activation of macrophages.
Abstract 660: Local Hemodynamic Forces and Aqueous Cigarette Smoke Extract Affect Development of Endothelial Dysfunction
Authors: Giebe Sindy , Brunssen Coy , Brux Melanie , Cockcroft Natalia , Hewitt Katherine , Morawietz Henning
Published: July 2021

Endothelial dysfunction is one of the first steps in the development of atherosclerosis. This proinflammatory phenotype is associated with decreased bioavailability of nitric oxide and a corresponding expression profile in the endothelial cells. Tobacco smoking promotes development of atherosclerotic plaques and local hemodynamic forces are key stimuli in this process. Low laminar flow is involved in the development of an unstable plaque phenotype, while high laminar flow has atheroprotective role. The molecular mechanisms controlling plaque stability in response to tobacco smoking remain largely unknown so far. Therefore, we exposed human endothelial cells to cigarette smoke extract (CSEaq) under disturbed flow conditions.

Primary human endothelial cells were stimulated with increasing dosages of CSEaq for 24h. Cell viability was reduced by CSEaq in a dose-dependent manner. The impact of specific flow conditions and different doses of CSEaq on the expression of atherosclerosis-related genes was investigated using a cone-and-plate viscometer. High laminar flow induced elongation of endothelial cells in the direction of flow, increased eNOS expression and NO release in a time-dependent manner. This increase was inhibited by CSEaq. Low laminar flow showed no effect on eNOS expression and NO release. The NRF2 antioxidative defense system was also induced by high laminar flow. NRF2 and NRF2 target genes HMOX1 and NQO1 were strongly activated by CSEaq. Furthermore, we monitored the expression of proinflammatory genes. CSEaq strongly induced adhesion molecule ICAM-1. Interestingly, VCAM-1 was unaffected by CSEaq. Induction of endothelial NADPH oxidase isoform 4 by CSEaq was prevented by high laminar flow. Catalase expression was not affected by flow and CSEaq, whereas CSEaq transiently increased SOD1 expression. Endothelial wound healing was improved by atheroprotective high laminar flow. Low flow did not affect wound healing. Furthermore, high laminar flow decreased adhesion of monocytes to endothelial cells, compared to low flow.

We suggest novel molecular mechanisms how tobacco smoking promotes the development of endothelial dysfunction. This can contribute to the formation of an unstable atherosclerotic plaque phenotype.
Abstract 673: KLF10 is a Critical Mediator of Wnt Signaling in Aortic Valve Interstitial Cells
Authors: Rajamannan Nalini M , Cicek Muzaffer , Hawse John , Spelsberg Thomas , Subramaniam Malayannan
Published: July 2021

We have previously demonstrated that β-catenin plays important roles in valve calcification with a specific osteogenic phenotype defined by increased bone mineral content and overall valve thickening. Recent studies indicate that KLF10 may be involved in mediating the Wnt signaling pathway in bone, which is known to play critical roles in osteoblast differentiation and mineralization. Therefore, we sought to test the role of KLF10 in mediating Wnt signaling, as well as differentiation and mineralization, in valve interstitial cells (VICs) isolated from porcine valves. Exposure of VICs to differentiation media led to increased expression of Runx2, Sox9 and osteocalcin. Differentiated cells also stained positive with Von Kossa while undifferentiated cells stained negative confirming the induction of an osteogenic phenotype. As expected, over-expression of both Lef1 and β-catenin led to activation of the top-flash reporter when transfected into VICs. Interestingly, over-expression of KLF10 also significantly up-regulated the top-flash reporter alone and further enhanced the activity of both Lef1 and β-catenin when co-transfected. We further confirmed the role of TIEG in an atherosclerotic mouse model ApoE/LRP5 double KO and found a two-fold increase in KLF10, Lrp6, and Runx2 expression in the cholesterol treated aortic valves as compared to controls. These data suggested that KLF10, Lef1 and β-catenin interact with each other to form a transcriptionally active protein complex leading to enhanced Wnt signaling in VICs. This possibility was further confirmed by the observation that KLF10 and β-catenin co-localize with one another in the nucleus of VICs following stimulation with LiCl and/or TGF-β. Taken together, these data implicate an important role for KLF10 in mediating Wnt signaling and Lef1 transcriptional activity in VICs, and implicate a potential role for canonical Wnt signaling in the observed osteogenic bone phenotype of cardiac aortic valves.
Abstract 110: The Separation of Serum Amyloid A from HDL is Mediated by Heparan Sulfate Through Histidine Dependent Interactions: HDL Function is Enhanced but Amyloid-Fibrils may be the “Price to Pay”¼
Authors: Ancsin John B , Munro Kim , Tam Shui-Pang , Davidson Michael H
Published: July 2021

Serum amyloid A (SAA) is an acute-phase protein that circulates bound to high density lipoprotein (HDL) and can influence HDL function as part of a poorly understood defense re-sponse to tissue trauma or infection. We have previously demonstrated that under mildly acidic pH the glycosaminoglycans, heparan sulfate (HS) and heparin can interact with HDL-SAA and cause SAA to dissociate from HDL. This remodeling improves HDL functionality but also predisposes SAA to form AA-amyloid fibrils. In this study we explore some potential pathophysiological conditions in vitro that could influence this HS/HDL-SAA remodeling process and the fate of SAA in vivo. SAA’s binding affinity for heparin was found to be enhanced by acidic pH and low concentrations of urea. The heparin dependent remodeling of HDL-SAA was promoted by the partial denaturation of HDL-SAA. Moreover, HDL-SAA remodeling was observed to follow a strict SAA:heparin stoichiometry and could be partially inhibited with a short heparin oligosaccharide of 8-sugar units. Evidence is also presented that once dissociated from HDL, SAA requires additional time to organize into Triton x-100 resistant amyloid-like structures. Circular dichroism spectroscopic analysis and in silico modeling of SAA’s ionizable residues highlights the importance of the histidine-36 within a highly conserved, pH-sensitive HS-binding site (HSBS-pH). A peptide containing the HSBS-pH sequence was demonstrated to have AA-amyloid seeding activity in a cell culture system. The recent determination of the 3D structure for human SAA1.1 has allowed the opportunity to re-assess and validate the HS/heparin binding sequences that had previously been identified biochemically with short synthetic peptides. We postulate that the dissociation of SAA from HDL takes place during the retro-endocytosis of HDL-SAA and is an important aspect of SAA function not previously appreciated.
Abstract 124: HDL Particles and HDL Functions
Authors: Asztalos Bela F , Horvath Katalin V , Goldfine Allison , Schaefer Ernst J
Published: July 2021

Introduction:
The role of HDL in CVD risk is not fully understood.

Hypothesis:
We tested the hypothesis that HDL functions are significantly associated with HDL subpopulation profile.

Methods:
Lipoproteins, including apoA-I-containing HDL particles, cell-cholesterol efflux (Global, ABCA1- and SRB1-mediated) and HDL Inflammatory Index were measured in a total of 289 subjects. These included 60 CHD patients, 109 subjects with either abnormal lipids (high triglycerides/low HDL or elevated β-sitosterol) or increased levels of the inflammatory markers C-reactive protein (CRP), serum amyloid A (SAA), fibrinogen, or myeloperoxidase (MPO), as well as 120 controls.

Results:
Global efflux was highly correlated with ABCA1-mediated efflux (r=0.921). Cholesterol efflux via the ABCA1 pathway was significantly correlated with small prebeta-1 HDL particles (r=0.762). CHD patients had significantly higher preβ-1 level than controls; however, per-particle prebeta-1 HDL effluxed 41% less cholesterol in CHD patients as compared to controls, resulting in no significant difference in overall efflux capacity between cases and controls. Cholesterol efflux via the SRB1 pathway was significantly correlated with large α-1 (r=0.784) and α-2 (r=0.460) HDL particles. These two large HDL particles effluxed about 82% of cell cholesterol via the SRB1 pathway. There was no significant difference in SRB1-mediated cholesterol efflux between CHD cases and controls. The HDL Inflammatory Index (HII) was significantly higher (unfavorable) in CHD cases than in controls and was significantly (inversely) associated with α-2 HDL particles (r=454). Increased levels of β-sitosterol significantly altered the HDL subpopulation profile and increased HII. Moreover, HII was not correlated with the concentrations of CRP, SAA, fibrinogen and MPO.

Conclusion:
HDL subpopulation profile is a major determinant of HDL’s overall cholesterol-efflux capacity. Efflux capacity of preβ-1 particles are compromised in CHD patients by yet unknown factor(s). HDL Inflammatory Index correlates with HDL subpopulation profile, but not with markers of inflammation in plasma.
Abstract 136: Identification of Melanoregulin as Novel Marker for Atherosclerosis
Authors: Aldi Silvia , Perisic Ljubica , Lengquist Mariette , Kronqvist Malin , Roy Joy , Bäcklund Alexandra , Ketelhuth Daniel F , Lindeman Jan H , Hansson Göran K , Paulsson-Berne Gabrielle , Hedin Ulf
Published: July 2021

Introduction:
Novel biomarkers identification in atherosclerosis is needed for early detection/intervention of vascular diseases. Impaired lysosomal function and autophagy cause abnormal lipid accumulation in foam cells and contribute to atherosclerosis progression. By microarray of human carotid plaques in the Biobank of Karolinska Endarterectomies (BiKE), we found that melanoregulin (MREG), a protein associated with lysosomes, phagocytosis and autophagy and previously not associated with atherosclerosis, was significantly up-regulated in symptomatic (S) versus asymptomatic (AS) patients. MREG expression was also significantly correlated to inflammatory, lipid and endo/lysosome markers.

Hypothesis:
We hypothesized that MREG modulates clearance of engulfed lipids and extracellular material in lipid-loaded foam cells.

Methods:
MREG mRNA and protein levels were evaluated in human carotid plaque tissue from S and AS patients by qPCR and immunohistochemistry, respectively. MREG, together with lysosome and lipid markers (LAMP2 and PLIN2 respectively) was identified by immunofluorescence in human monocyte cells (THP-1), loaded with modified LDL. To investigate autophagy/MREG correlation, THP-1 cells were incubated with autophagy inhibitor (chloroquine, CQ) and MREG mRNA was assessed by qPCR.

Results:
MREG mRNA is elevated in human atherosclerotic lesions versus normal artery (11.21 folds, P<0.0001). In human aortic tissue MREG expression is correlated to the lesion progression and localizes in infiltrating macrophage-derived foam cells. In the human carotid plaque, MREG co-localizes with CD68+ and PLIN2+ cells, lined to the necrotic core. MREG protein was also detected in PMA-differentiated THP-1 cells. In lipid-loaded THP-1, MREG is co-localized with lysosomal LAMP2. Moreover, MREG expression is increased in THP-1 cells by incubation with PMA, starving medium and CQ.

Conclusions:
We show here for the first time that MREG is expressed in atherosclerotic lesions and is associated with endo/lysosomes in human macrophages. Furthermore we show that autophagy inhibition increases MREG mRNA expression. Future studies will investigate how MREG expression levels will affect phagocytic activity of lipid-loaded macrophages.
Abstract 139: Vascular Endothelin-1 Activity Increases with Age in Hypertensive Patients
Authors: Berdy Andrew E , Brar Vijaywant , Panza Julio A , Campia Umberto
Published: July 2021

Background:
Vascular endothelin-1 (ET-1) dependent vasoconstriction is elevated in patients with hypertension. The effect of increasing age on ET-1 activity is unknown. This study investigated the hypothesis that age is directly associated with ET-1 activity in hypertensive patients.

Methods:
Retrospective analysis of data collected in a prospective clinical study that investigated vascular ET-1 activity in patients with essential hypertension (BP>140/90mmHg). Endothelin-1 activity was expressed as forearm blood flow (FBF) responses as measured by strain-gauge plethysmography during the intra-arterial infusion of the selective endothelin type-A receptor antagonist BQ-123 (100 nmol/min) for 60 minutes. Changes in FBF from baseline in response to BQ-123 were assessed by one-way ANOVA for repeated measures. Association analyses were performed with use of the Pearson correlation coefficient.

Results:
Data from 40 patients were included in the current analyses. The baseline characteristics of the study population are reported in the table. Infusion of BQ-123 induced a significant increase in FBF from baseline (p<0.001). The percent increase in FBF from baseline at 60 minutes of BQ-123 infusion was significantly directly associated with age (r=0.435; p=0.006), but not with blood pressure, parameters of glucose and lipid metabolism, markers of endothelial activation, and adipocytokine levels (p=NS for all).

Conclusions:
In patients with essential hypertension, ET-1 activity is directly associated with age. Our findings suggest that, in this population, aging may contribute to vascular damage and cardiovascular disease risk through an enhanced activation of the ET-1 system. Further study is needed to elucidate the mechanisms underlying the association between age and vascular ET-1 activity.
Abstract 143: Macropinocytosis Mediates Enzymatically Modified LDL (ELDL)-Induced Murine Smooth Muscle Cell Formation: A Role for RAGE In ELDL Endocytosis and Upregulation of Scavenger Receptor LOX1
Authors: Chellan Bijoy , Reardon Catherine A , Hofmann Bowman Marion
Published: July 2021

Background:
ELDL is present in human atherosclerotic lesions and promotes foam cell formation in cultured macrophages and vascular smooth muscle cells (SMC). Here we study mechanism of ELDL uptake and its effects on SMCs.

Methods and Results:
Incubation of wild type murine aortic SMCs with 10 μg/ml ELDL (trypsin, cholesterol esterase modified) results in enhanced foam cell formation (analyzed by Oil Red O, lipid measurement) compared to SMCs incubated with acetylated LDL (500 μg/ml; -50%, p<0.01) and oxidized LDL (200 μg/ml; -75%, p<0.01). Inhibitors of macropinocytosis (50 μM LY294006, 2 μM wortmannin, and 3 mM amiloride) attenuated ELDL uptake (-50%, -50%, -100% respectively). In contrast, inhibitors of receptor mediated endocytosis (100 μM dynasore, 0.1 M Sucrose), and inhibitors of caveolae /lipid raft mediated endocytosis (5mM MBCD, 5 μM filipin) had no effect on ELDL uptake in SMCs. Moreover, ELDL incubation led to increased expression of scavenger receptor LOX1 (+ 3 fold, p<0.01) in wild type SMC’s, but not in SMC deficient in Receptor for AGE (RAGE-/-), while CD36 and SRA1 remained unchanged in both the SMCs. Importantly, RAGE-/- SMCs upon pretreatment with PI3K inhibitors that only partially inhibited macropinocytosis of ELDL in wild type SMCs, completely prevented ELDL uptake in RAGE-/- SMCs. Mechanistically, ELDL upregulates ROS (detected using H2DCFDA) and down regulates PIP3 (detected by pAkt immunoblotting) in wild type, but not in RAGE-/- SMCs. Since ROS is known to regulate macropinocytosis via increased Ca2+ levels, we tested Ca2+ channel inhibitor lacidipine (30 μM), and found complete inhibition of ELDL uptake in both, wild type and RAGE-/- SMCs. Lastly, we speculate that the fused structure of LDL in the ELDL preparation is preferentially activating RAGE, since oligomerization of ligands are known to increase RAGE signaling, and FPLC analysis demonstrated that ELDL consists mostly of fused LDL particles.

Conclusions:
ELDL is highly potent in inducing foam cells in aortic SMCs. ELDL endocytosis is mediated by RAGE-regulated, Ca2+ dependent macropinocytosis.
Abstract 152: A Novel Targeted Proteomics Approach Discovers Biomarkers for Human Atherosclerosis
Authors: Ingelsson Erik , Ärnlöv Johan , Lindahl Bertil , Siegbahn Agneta , Sundström Johan , Lind Lars
Published: July 2021

Background:
We used a proteomics array to simultaneously measure 92 proteins that have been suggested to be associated with atherosclerosis and related them to plaque prevalence and echogenicity in carotid arteries in a human population-based study.

Methods:
In the Prospective Study of the Vasculature in Uppsala Seniors (PIVUS; n=931, 50% women, all aged 70 years), the number of carotid arteries with plaques was recorded by ultrasound and plaque echogenicity was determined. Levels of 92 proteins were assessed in plasma by a proximity extension assay (Proseek Multiplex CVD, Olink Bioscience, Uppsala, Sweden) and related to carotid measures in a regression framework.

Results:
Following adjustment for multiple testing, six of the proteins were significantly related to the number of carotid arteries affected by plaques in sex-adjusted models (osteoprotegrin, T-cell immunoglobulin and mucin domain-1, renin, growth hormone, matrix metalloprotease-12 and lecitin-like oxidized LDL receptor 1). When these six proteins were included jointly in a model adjusting for traditional cardiovascular risk factors, renin (odds ratio [OR], 1.30; 95% confidence interval [CI], 1.13-1.50 per standard deviation increase) and growth hormone (OR, 1.26; 95% CI, 1.10-1.45) were related to plaque prevalence independently of each other and known risk factors for atherosclerosis. Thirty-one proteins were related to plaque echogenicity after adjustment for known risk factors. Of these, interleukin-27 subunit alpha (beta, 5.20; P=0.00032), vascular endothelial growth factor-delta (beta, 4.73; P=0.0013) and matrix metalloprotease-1 (beta, 3.93; P=0.0034) were associated with plaque echogenicity independently of each other.

Conclusion:
A novel targeted proteomics approach using the proximity extension technique discovered several new associations of candidate proteins with plaque prevalence and echogenicity in the carotid arteries in a large human sample.
Abstract 168: The Acceleration of Foam Cell Formation by Chlamydia Pneumoniae Requires Nlrp3 Inflammasome Activation and Il-1 Signaling
Authors: Tumurkhuu Gantsetseg , Dagvadorj Jargalsaikhan , Crother Timothy R , Shimada Kenichi , Arditi Moshe , Chen Shuang
Published: July 2021

Background & Objective:
Foam cell formation (FCF) due to excessive accumulation of cholesterol by macrophages is a pathological hallmark of atherosclerosis. Chlamydia pneumoniae (Cp) promotes FCF in the presence of oxLDL, but the exact molecular mechanisms are still not completely delineated. Recent data indicates that the Nlrp3 inflammasome plays an important role in the formation of atherosclerotic plaques. Here we investigated the role of the Nlrp3 inflammasome during the acceleration of FCF by Cp infection.

Methods and Results:
In order to determine if the NLRP3 inflammasome played a role in Cp infection induced acceleration of FCF, we treated resident peritoneal macrophages exposed to oxLDL and Cp with the IL-1R antagonist, Anakinra, to block IL-1 signaling. Treatment with Anakinra resulted in a significant reduction in FCF. Nlrp3-/-, Casp1-/-, and WT macrophages were also treated with live Cp in the presence or absence of oxLDL. We found that Nlrp3-/- and Casp1-/- macrophages had significantly less FCF compared with WT cells. Interestingly, both ABCA1 (cholesterol efflux transporter) and its transcription factor, liver X receptor (LXR-α), were increased in Nlrp3-/- and Casp1-/- macrophages compared with WT cells. Addition of rIL-1β to Nlrp3-/- macrophages led to a decrease in ABCA1 expression and greater FCF. Importantly, Il1r-/- macrophages also had greater ABCA1 expression and reduced FCF when exposed to oxLDL and Cp infection.

Conclusion:
These data suggest that Cp infection facilitates foam cell formation in the presence of oxLDL by producing NLRP3 dependent IL-1 cytokines, which then feed back on the macrophages and interferes with cholesterol efflux by negatively regulating ABCA1. In the absence of IL-1 signaling, the expression of ABCA1 is upregulated leading to greater cholesterol efflux and reduced FCF. Thus we have identified a novel regulatory loop controlling FCF. Understanding these interacting pathways will lead to new therapeutic strategies against atherosclerosis.