bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 538: Metabolism of Multiple Apolipoproteins Across HDL Size in Humans
Authors: Andraski Allison B , Singh Sasha A , Pieper Brett , Goh Wilson , Mendivil Carlos O , Aikawa Masanori , Sacks Frank M
Published: June 2021

Introduction:
Studying the kinetics of several HDL apolipoproteins together may help elucidate their function in HDL metabolism. We present multicompartmental analysis describing the metabolism of several apolipoproteins across 5 HDL size fractions.

Methods and Results:
Three participants were infused with a bolus of D3-Leu tracer, and blood was collected for 70 hrs. ApoA-I-HDL was prepared by immunoaffinity purification, separated into 5 size fractions, preβ, α3, α2, α1, and α0, by ND-PAGE, and in-gel trypsinized for mass spectrometry. We monitored 7 proteins that likely affect HDL metabolism - apoA-I, apoA-II, apoE, apoM, apoC-III, apoA-IV, and apoD. We used stable isotope labeled peptide standards to quantify pool size and high resolution parallel reaction monitoring to measure tracer enrichment in these 7 proteins in the 5 HDL size fractions. These data were then used for multicompartmental analysis to describe the kinetic behaviors across HDL size for each apolipoprotein, with the exception of apoD. All size fractions with discernible enrichment curves for at least 2 participants were included in the final model for each apolipoprotein (Fig.1). The majority of apoA-I α HDL originated form the source compartment, presumably the liver and/or small intestine, while apoA-I preβ HDL came primarily from α3. Size expansion pathways (preβ to α1/2, and α3 to α2) contributed only slightly to apoA-I metabolism. Similarly to apoA-I on the α sizes, the majority of the other apolipoproteins appear on each HDL size fraction directly from the source compartment. Only minor flux pathways from smaller to larger HDL sizes were identified: apoA-II, α3 to α2; apoE, α3 to α2, and α3 to α1.

Conclusions:
This study supports the new model of HDL metabolism in which HDL and its attached apolipoproteins are metabolized mainly within each HDL size fraction. Pathways between HDL sizes only provide a small contribution to the metabolism of each apolipoprotein.
Abstract 54: Differential Regulation of NADPH-oxidases 1 and 2 and a Common Syk/PLC/Calcium-dependent ROS Signaling Pathway Mediating Platelet Activation
Authors: Xu Zheng
Published: June 2021

Objective:
Reactive oxygen species (ROS) generated from activated platelets is known to regulate platelet activation. This study investigates how different isoforms of nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) oxidases (NOXs) mediates different platelet activation pathways.

Approach and Results:
ROS generation in different platelet activation pathways are mediated differentially by NOX1 and NOX2. NOX1
-/y
platelets showed no defects in platelet aggregation and secretion induced by glycoprotein (GP) VI agonists, collagen-related peptide (CRP), but were partially defective in platelet aggregation and secretion induced by low doses of agonists of G protein coupled receptor (GPCR), thrombin, protease-activated receptor 4 agonist peptide (PAR4AP) and thromboxane A2 analog U46619. In contrast, NOX2
-/-
platelets showed significantly defective platelet aggregation and secretion induced by CRP, and also showed partial inhibition of thrombin-induced platelet aggregation and secretion. Consistently, production of reactive oxygen species (ROS) was inhibited in NOX1
-/-
platelets stimulated with thrombin, but not CRP, whereas NOX2
-/-
platelets were defective ROS generation induced by CRP or thrombin. These differential effects of NOX1 and NOX2 is likely due to upstream differential regulation of these different enzymes, as thrombin-stimulated NOX1-/y platelets and CRP-stimulated NOX2-/- platelets similarly showed defective activation of tyrosine kinase Syk, its downstream target phospholipase Cγ (PLCγ) and calcium mobilization, which is mediated by PLC. Furthermore, mitogen-activated protein kinase pathways, which is another important platelet activation pathway was not significantly affected in either NOX1-/y or NOX2-/- platelets. Finally, NOX-/- platelets is defective in mediating arteriolar thrombosis in vivo, although minimally affected tail bleeding time.

Conclusions:
NOX1 and NOX2 play differential roles in different platelet activation pathways. The differential roles of these enzyme are due to differential upstream regulation. Both NOX isoforms mediates platelet activation via a common ROS-dependent activation Src-PLC-calcium signaling pathway.
Abstract 541: The Molecular Interaction of Apolipoprotein A-I Containing High Density Lipoproteins with Lecithin: Cholesterol Acyl Transferase
Authors: Cooke Allison , Melchior John T , Morris Jamie C , Huang Rong , Jerome W. Gray , Davidson W. Sean
Published: June 2021

The structure of apolipoprotein (apo)A-I on a high density lipoprotein (HDL) particle can elucidate the protein interactions and cardioprotective functions of HDL. This includes critical interactions with HDL modifying proteins like lecithin:cholesterol acyl transferase (LCAT) which performs a key function in reverse cholesterol transport by using apoA-I as a cofactor to esterify cholesterol. Data from our lab and others demonstrate that apoA-I molecules dimerize into an antiparallel stacked ring structure that encapsulates lipid in reconstituted (r)HDL particles. Cross-linking analysis of rHDL particles imply two possible registries: one with the fifth helical repeat of an apoA-I molecule adjacent to the fifth helical repeat of its antiparallel partner (5/5), and the other with the fifth helical repeat adjacent to the second helical repeat of its antiparallel partner (5/2). We hypothesized that apoA-I registry on rHDL can modulate LCAT activity. Site-directed cysteine mutagenesis was used to lock two apoA-I molecules into each of the aforementioned helical registries, and an intermediate registry created as a negative control. rHDL particles were generated using these dimerized mutants, and their ability to activate LCAT was determined. The 5/5 mutant demonstrated higher LCAT activity than wild-type rHDL particles while the 5/2 and intermediate mutants showed dramatically lower LCAT activity (p<0.001). To determine where LCAT interacts with apoA-I, rHDL containing wild-type apoA-I was cross-linked to LCAT. The majority of the cross-links were concentrated between S240 of LCAT and a region encompassing helices 4-6 of apoA-I. We propose that LCAT binds to a discontinuous epitope comprised of two apoA-I molecules in rHDL and that changes in this registry can alter LCAT function. These studies provide a basis for understanding how apoA-I structure may modulate the association and activity of a multitude of different HDL protein partners. This implicates apoA-I conformation as a target for altering HDL function in order to enhance the cardioprotective properties of HDL.
Abstract 550: TFEB Expression, Turnover, and Nuclear Localization are Altered in DBA/2 Mouse Macrophages Associated With Impaired Autolysosome Formation and Lipid Droplet Clearance
Authors: Robinet Peggy , Smith Jonathan D
Published: June 2021

In a previous study, we identified autolysosome formation as the limiting step for turnover of cholesterol esters in lipid droplets of macrophage foam cells from the atherosclerosis sensitive DBA/2 strain compared to the atherosclerosis resistant AKR mouse strain. As autophagosome formation was similar in these two strains, we wanted to evaluate the role of lysosomes in autolysosome formation in AKR and DBA/2 cells. For all our studies, we cultured AKR and DBA/2 bone marrow-derived macrophages with or without acetylated LDL (AcLDL) for 24h. Lysosome function and number, assessed by flow cytometry on Lysotracker red DND-99 (LyT) labeled cells, was not affected by AcLDL loading in AKR and DBA/2 cells. However, in both unloaded and loaded conditions, DBA/2 cells exhibited a 30 to 50% lower LyT intensity suggesting that they have intrinsically decreased lysosome number/function. Lysosomal degradation capacity was assayed by incubation with DQ-ovalbumin and we observed a 27% decrease in lysosome function in DBA/2 vs. AKR foam cells (p<0.01). The impairment of lysosomal degradation was confirmed by 44.8% decreased levels of cathepsin L mature form in DBA/2 vs. AKR foam cells (p<0.05). As the transcription factor TFEB is a key regulator for lysosome biogenesis and function, we studied this factor in our system. First, we analyzed TFEB protein expression by western blot. Upon cholesterol loading, TFEB was induced in AKR (48%, p<0.01) but not DBA/2 cells leading to a 45% higher TFEB levels in AKR vs. DBA/2 foam cells (p<0.05). A preliminary evaluation of TFEB turnover by western blot also revealed that, upon loading, TFEB half-life is twice as long in AKR vs. DBA/2 macrophages (10.1h and 4.8h, respectively). Finally, we investigated TFEB nuclear translocation by immunofluorescence. AKR and DBA/2 macrophages were labeled with an antibody against TFEB and the percentage of TFEB positive nuclei was assessed. After 24h AcLDL loading, only 26% of DBA/2 nuclei contained TFEB vs. 48% for AKR nuclei (p=0.0002 by Fisher’s exact test). In conclusion, we found that DBA/2 vs. AKR foam cells have altered TFEB processing that may explain the altered lysosome number and function. This may result in the autolysosome formation defect in the DBA/2 foam cells.
Abstract 552: End Stage Human Aneurysm Disease in Different Arterial Positions is Similar, Aneurysm Induction in Mouse Models is Not
Authors: Busch Albert , Schmidt Caroline , Paloschi Valentina , Eriksson Per , Ergün Süleyman , Lorenz Udo , Hartmann Elena , Kellersmann Richard , Maegdefessel Lars , Otto Christoph
Published: June 2021

Introduction:
Aneurysm disease occurs throughout the arterial tree, abdominal aortic aneurysm (AAA) and popliteal artery aneurysm (PAA) being the most frequent ones. The pathology is best examined, yet little understood, for AAA. Aneurysm disease has distinct features like angiogenesis and phenotype switch of vascular smooth muscle cells (VSMC). However, little is known about these characteristics in other than AAA.

Material and Methods:
From a surgical biobank we compared 42 AAA, 15 PAA, 3 ascending aortic, 5 iliac, 3 femoral, 2 brachial, 1 visceral and 1 carotid artery aneurysm on morphologic, protein and mRNA-expression levels. Two multi-stage mouse models of aneurysm disease were applied to test response to aneurysm induction at different arterial locations.

Results:
AAAs show a varying histomorphologic appearance, not seen in PAA, which, in turn, has a high proliferation rate in the aneurysm sac, making other than surgical interventions appealing. All entities show VSMC phenotype switch, angiogenesis, matrix remodeling and T-cell/macrophage inflammation. AAA and PAA have similar alterations in TGFß-signaling. To further investigate these conditions in mouse models, we applied the PPE procedure to a juxtarenal aorta and the EPA model to a thoracic, abdominal and femoral location.

Conclusion:
Despite different arterial morphogenesis, advanced aneurysm disease from human intraoperative specimen shows similar characteristics of end-stage disease.
Abstract 559: Lectin-like Oxidised Low Density Lipoprotein Receptor-1 Expression in Atheroma of Patients with Coronary Artery Disease is Higher in Patients with Acute Coronary Syndrome but Decreased in Patients on Statins
Authors: Landsberger Martin , Ackermann Jens , Felix Stephan B , Dahm Johannes B
Published: June 2021

Introduction:
The lectin-like oxidised low density lipoprotein receptor-1, LOX-1, is expressed in endothelial cells and macrophages among others. Its expression can be induced by a variety of stimuli such as oxidised LDL, endothelin-1, angiotensin II and shear stress. LOX-1 seems to be important for the induction of endothelial dysfunction and is implicated in atherosclerotic plaque vulnerability.

Methods:
Atherectomy specimens were obtained from 37 patients (mean age 62.5±9.7 years, body mass index 28.6±5.3 kg/m2; n=30 with chest pain/angina pectoris), who underwent medially indicated directional coronary atherectomy in the Department of Internal Medicine B, University Medicine Greifswald, between July 2001 and April 2005, and were immediately frozen in liquid nitrogen. Human coronary atherectomy specimens were cut into 5 μm-slices using a kryotom and stained with haematoxylin and eosin (H&E), Oil Red O (lipids) and Elastica-van Gieson (collagen). LOX-1 was detected by immunofluorescence using a FITC-labelled rabbit polyclonal antibody against amino acids 143 to 271 of human LOX-1. Digital images were analysed with Corel PHOTO-PAINT 12, SCION Image, and SigmaPlot 11. Correlations between signal intensities for LOX-1 expression and patients’ parameters were analysed by Spearman Rank Order testing. Patients gave informed consent. The study protocol complied with the Declaration of Helsinki and was approved by the local ethics committee.

Results:
LOX-1 expression correlated significantly with lipid content of the coronary atherectomy specimens. LOX-1 expression tended to be higher in specimens from patients with acute coronary syndrome (n=11) and to be lower in patients receiving statins (n=16). However, no correlations were found between LOX-1 expression and grade of stenosis, plaque localisation, age, sex, body mass index, smoking (n=15), hypertension (n=31), diabetes mellitus type II (n=9), and dyslipoproteinaemia (n=32).

Conclusions:
LOX-1 expression in plaques was positively correlated with lipid contents of plaque material and tended to be higher in patients with acute coronary syndrome and to be lower if patients had received statins. High LOX-1 expression in the atherosclerotic plaque may contribute to plaque instability.
Abstract 56: Coagulation Factor XI Promotes Distal Platelet Activation and Single Platelet Consumption in the Bloodstream Under Shear Flow
Authors: Zilberman-Rudenko Jenya , Wiesenekker Chantal , Itakura Asako , McCarty Owen J
Published: June 2021

Objective:
Coagulation factor XI (FXI) has been shown to contribute to thrombus formation on collagen or tissue factor (TF)-coated surfaces
in vitro
and
in vivo
by enhancing thrombin generation. Whether the role of the intrinsic pathway of coagulation is restricted to the local site of thrombus formation is unknown. This study was designed to determine whether FXI could promote both proximal and distal platelet activation and aggregate formation in the bloodstream.

Approach and Results:
Pharmacological blockade of FXI activation or thrombin activity in blood did not affect local platelet adhesion, yet reduced local platelet aggregation, thrombin localization and fibrin formation on immobilized collagen and TF under shear flow,
ex vivo
. Downstream of the thrombus formed on immobilized collagen or collagen and 10 pM TF, platelet CD62P expression and microaggregate formation and progressive platelet consumption were significantly reduced in the presence of FXI-function blocking antibodies or a thrombin inhibitor in a shear rate- and time-dependent manner. In a non-human primate model of thrombus formation, we found that inhibition of FXI reduced single platelet consumption in the bloodstream distal to a site of thrombus formation.

Conclusions:
This study demonstrates that the FXI-thrombin axis contributes to distal platelet activation and procoagulant microaggregate formation in the blood flow downstream of the site of thrombus formation. Our data highlights FXI as a novel therapeutic target for inhibiting distal platelet activation without affecting proximal platelet adhesion.
Abstract 562: Impaired Transcriptional Regulation of Nrf2 and Antioxidant Signaling in Vascular Tissue of Aging Mice
Authors: Radhakrishnan Rajesh Kumar , Shanmugam Gobinath , Narasimhan Madhusudhanan , Devarajan Asokan , Davidson Christopher , Namakkal Soorappan Rajasekaran
Published: June 2021

Background:
Chronic oxidative stress and dysregulation of vascular system are tightly linked to cardiac structure-function anomalies. We hypothesized that nuclear erythroid-2 like factor-2 (Nrf2), a redox-sensitive transcriptional regulator, distinctly regulate antioxidant transcription in aortic vessels on aging versus high intensity exercise stress (HIES).

Methods:
Age and sex matched wild-type (WT) and Nrf2
-/-
mice at young (3 months) and old (>20 months) ages were used in this study. Effect of loss of function for Nrf2 on transcription and translation of targets involve in antioxidant signaling was studied in response to HIES on aging.

Results:
Under basal setting, Nrf2 abrogation resulted in moderate downregulation of a subset of antioxidant gene expression (
G6pd, catalase, Gclc, Gclm, Gstα, Gstμ,
and
Nqo1
) in aortic vessel in relation to age-matched wild-type (WT) mice. Interestingly, some of the antioxidant genes (
G6pd, Sod1, Sod2, Ho1, Gclc, Gclm, Gstα
and
Gstμ
) were significantly downregulated in old-WT when compared to young-WT mice, suggesting age-associated impairment of Nrf2-dependent antioxidant signaling. On the other hand, the expression of certain antioxidant genes (
Gclm, Catalase, Gsr, Nqo1, Gpx1
and
Gstμ
) were similar in HIES-subjected WT and Nrf2
-/-
mice in relation to sedentary controls at old age. These observations suggest that the response to HIES is blunted on aging due to either impairment or ablation of Nrf2 in WT versus knockout mice. In addition, we observed a significant increase in mRNA levels of Keap1, G6pd, Sod1 and Sod2 in aorta of Nrf2
-/-
mice when compared to its WT counterparts. These results indicate that either abrogation (in Nrf2
-/-
mice) or age-associated decline (in old WT mice) of Nrf2 describes a distinctive molecular phenotype related to transcriptional regulation of redox signaling on aging.

Conclusion:
Thus, our findings direct that dysregulation and/or absence of Nrf2 could impair the physiological adaptive events of aortic vessel to intense exercise stress tending to pathologic vascular remodeling and may therefore result in cardiovascular complications.
Abstract 563: Oxidized LDL Promotes Endothelial NF-kappaB Activation and Inflammation Through Focal Adhesion Kinase-dependent RSK Signaling
Authors: Yurdagul Arif , Pattillo Christopher B , Schlaepfer David D , Orr Wayne
Published: June 2021

Oxidized LDL (oxLDL) accumulates early in atherosclerosis and promotes endothelial NF-kappaB activation, proinflammatory gene expression, and monocyte adhesion. Like other atherogenic factors, oxLDL-induced proinflammatory responses requires integrin-dependent focal adhesion kinase (FAK) signaling; however, the mechanism by which FAK mediates oxLDL-dependent NF-kappaB signaling has yet to be revealed. We now show that oxLDL induces NF-kappaB activation and VCAM-1 expression through FAK-dependent IkappaB kinase beta (IKKbeta) activation. We further identify FAK-dependent activation of p90 ribosomal S6-kinase (RSK) as a critical mediator of oxLDL-dependent IKKbeta/NF-kappaB signaling, as inhibiting RSK blocks oxLDL-induced IKKbeta/NF-kappaB activation, VCAM-1 expression, and monocyte adhesion. Lastly, transgenic mice containing a kinase-dead mutation in FAK specifically in the endothelial cells show reduced RSK activity, decreased VCAM-1 expression, and reduced macrophage accumulation in regions of early atherosclerosis. Taken together, our data elucidates a novel mechanism whereby oxLDL-induced endothelial FAK signaling drives an ERK/RSK pathway to activate IKKbeta/NF-kappaB signaling and proinflammatory gene expression.
Abstract 564: Inflammasome-induced Endothelial Microparticles Exert Detrimental Effects on Recipient Vascular Cells
Authors: Pfeifer Philipp , Ebert Sophia-Katharina , Liu Lin , Jansen Felix , Jehle Julian , Latz Eicke , Franklin Bernardo S , Nickenig Gerog , Werner Nikos
Published: June 2021

Background:
The inflammasome, a multi-protein signaling platform, is an important mediator of vascular inflammation resulting in atherosclerosis. Microparticles (MPs) are small membrane vesicles, specifically packaged and released from apoptotic cells for intercellular communication. Endothelial cell derived MPs have recently been linked to atherogenesis. Whether inflammasome activation in endothelial cells mediates MP release and thereby exert biological effects on recipient vascular cells is unknown.

Methods and results:
Human coronary artery endothelial cells (HCAEC) were primed with 1μg/ml LPS and subsequently stimulated with 20μM Nigericin leading to a specific inflammasome activation as detected by significant upregulation of Caspase-1 mRNA in RT-PCR and protein in Westernblots (Figure1). Inflammasome activation in vascular cells lead to formation of endothelial microparticles (EMP) in a time- and dosis-dependend manner (Figure 2). Comparison of EMP with beads of known size using flow cytometric and electron microscopic imaging could point out an EMP-size between 0,1 – 1 μm (Figure 3). EMP-uptake by recipient vascular cells could be illustrated by fluorescence-microscopic imaging of PKH26 labeled EMP (Figure 4). Viability assay and scratch assay showed detrimental effects of EMPs from inflammasome activated cells on recipient vascular cells. Viability, proliferation and migration were reduced significantly 4h after treatment (Figure 5).

Conclusions:
We show for the first time that Nigericin, an established inflammasome activator, leads to inflammasome activation and release of microparticles by endothelial cells. Furthermore, that these microparticles are taken up by recipient vascular cells and thereby cause cell death accompanied with reduced cell migration and proliferation.
Abstract 57: Podoplanin-clec-2-mediated Platelet Release Protects Vascular Stability in the Mouse Developing Brain
Authors: Fu Jianxin , Xia Lijun
Published: June 2021

Angiogenesis in the central nervous system (CNS), driven by neural cell-derived vascular endothelial cell growth factor (
VEGF
)
, is extremely active during early embryonic development in mice (E10.5 - 13.5). The blood brain barrier (BBB) is not fully functional during this stage. It is unclear whether there isadditional unidentified mechanism(s) protect the integrity of the nascent vessels in the developing brain. Podoplanin (PDPN) is highly expressed in neural cells between E9.5 - 14.5. We found that mice lacking PDPN develop spontaneous bleeding in the developing CNS. Furthermore, mice with specific deletion of PDPN on neural progenitor cells or its receptor CLEC-2 on platelets developed similar brain bleeding phenotype, supporting that interactions of neural PDPN with platelet CLEC-2 are critical for vascular stability during development. We identified platelet(s) at the abluminal side of vessels in the WT embryonic brain in close proximity to PDPN expressing neural cells, supporting that extravasated platelets interact with perivascular neural cells and are activated through CLEC-2 signaling pathway. Furthermore, loss of PDPN or CLEC-2 leads to decreased endothelial intercellular junctional protein, VE-cadherin. In vitro analysis demonstrated that PDPN-CLEC-2-mediated platelet release counteracts the effects of VEGF on VE-cadherin expression and blood vessel permeability. These results provide a novel mechanism for the protection of nascent vessels in the developing brain.
Abstract 570: Cystathionine γ-Lyase Modulates Flow-dependent Vascular Remodeling
Authors: Yuan Shuai , Yurdagul Arif , Green Jonette M , Pardue Sibile , Kevil Christopher G , Orr A. Wayne
Published: June 2021

Disturbed flow causes endothelial dysfunction and development of atherosclerotic lesions. The gaseous signaling molecule H
2
S and cystathionine γ-lyase (CSE), its major enzymatic source in the vasculature, protect against cardiovascular diseases including atherosclerosis, peripheral artery disease, and cardiac ischemia in a nitric oxide (NO) dependent manner. Therefore, we sought to investigate the role of CSE in the endothelial response to disturbed flow. Wild-type C57Bl/6 (WT) and CSE knockout (CSE-/-) mice underwent partial carotid ligation to induce disturbed flow in the left carotid with the right carotid serving as an internal control. Additionally, endothelial cells isolated from WT and CSE-/- mice were exposed to oscillatory flow, a model of disturbed flow, in vitro. While disturbed flow decreased endothelial CSE mRNA expression, CSE protein expression showed strong induction under disturbed flow conditions both in vitro and in vivo. This induction correlated with enhanced free sulfide and sulfane sulfur production in WT but not in CSE-/- mice. Intimal mRNA isolated 2 days post-ligation showed increased VCAM-1 and ICAM-1 expression in WT mice which was prevented in CSE-/- mice. Similarly, endothelial cells isolated from CSE-/- mice show reduced NF-B activation and proinflammatory gene expression in response to oscillatory flow in vitro. Morphometric analysis of carotid arteries collected 7 days post-ligation revealed reduced macrophage infiltration and medial thickening in the ligated carotid of CSE-/- mice. Interestingly, ligation increased the carotid nitrite level in WT mice but not in CSE-/- mice. However, nitrite level of the non-ligated carotid was significantly higher in the CSE-/- mice compared to WT mice. Shear induced phosphorylation of eNOS Ser1179 in vitro was not different between WT and CSE knockout endothelial cells, suggesting alternative regulatory mechanisms. Collectively, CSE in mouse carotid arteries plays a critical role in flow dependent vascular remodeling, which may be mediated by CSE derived free sulfide and sulfane sulfur. CSE deficiency completely inhibits disturbed flow-induced NF-κB activation and macrophage recruitment, consistent with the role of inflammation in vascular remodeling.
Abstract 579: Graft-Derived IL-6 Amplifies Proliferation and Survival of Effector T Cells that Drive Alloimmune-Mediated Vascular Rejection
Authors: von Rossum Anna , Rey Kevin , Enns Winnie , Cheema Rajan , MacEwan Grace E , Manku Sukh , Choy Jonathan C
Published: June 2021

Objective:
IL-6 is an inflammatory cytokine that controls the development of effector T cell responses but the mechanisms by which it controls allogeneic immune responses and vascular rejection that leads to transplant arteriosclerosis (TA) is poorly understood. We have examined the effect of graft-derived IL-6.

Methods:
An aortic interposition model of vascular rejection and TA was performed using IL-6
+/+
and IL-6
-/-
donors.

Findings:
The absence of IL-6 production from artery graft cells reduced the development of vascular rejection and arteriosclerotic thickening. There was no apparent effect of donor-derived IL-6 on endothelial cell integrity or on the intimal accumulation of smooth muscle cells and macrophages. However, reduced vascular pathology in IL-6
-/-
artery grafts was accompanied by a significant reduction in the accumulation of CD4 and CD8 T cells. Further, the absence of graft-derived IL-6 resulted in a significant decrease in the activation and proliferation of alloreactive CD4 and CD8 T cells after transplantation as well as in a marked increase in cell death of effector T cells. Alloreactive effector T cells that developed in the absence of IL-6 were also more susceptible to Fas-mediated activation-induced cell death
in vitro
.

Conclusions:
Donor-derived IL-6 amplifies the expansion of allogeneic T cell responses that cause vascular rejection and TA by increasing T cell proliferation and preventing Fas-mediated T cell death.
Abstract 584: Smooth Muscle Cell-derived IL-17C Plays an Atherogenic Role via the Recruitment of Pro-inflammatory IL-17A
+
T Cells to the Aorta
Authors: Butcher Matthew J , Waseem Tayab C , Galkina Elena V
Published: June 2021

Atherosclerosis is characterized by frequent communication between infiltrating leukocytes and vascular cells, through chemokine and cytokine networks. IL-17 cytokine family members, including IL-17C, are detectable within atherosclerotic plaques, however the potential involvement of these cytokines have not been examined. Thus we sought to investigate the role of IL-17C in atherosclerosis. The expression of IL-17 cytokines was profiled within atherosclerotic

Apoe
-/-

aortas and
Il17c
expression was elevated. Flow cytometry experiments revealed a major population of aortic IL-17C-producing smooth muscle cells. To determine the role of IL-17C in atherosclerosis, we generated

Il17c
-/-
Apoe
-/-

mice and compared atherosclerotic lesions between western diet-fed

Apoe
-/-

and

Il17c
-/-
Apoe
-/-

mice. Atherosclerotic lesion and collagen content was diminished within WD-fed

Il17c
-/-
Apoe
-/-

aortas and aortic roots in comparison to

Apoe
-/-

controls, and IL-17C treated

Apoe
-/-

aortas up-regulated Col1A1 expression
ex vivo
. Flow cytometric analysis of

Il17c
-/-
Apoe
-/-

aortas revealed a proportional reduction in aortic leukocytes, macrophages, neutrophils, T cells, Th1, and T regulatory cells, without corresponding changes in the peripheral immune composition. Examination of aortic IL-17A
+
TCRγδ T cells and Th17 cells demonstrated a stark reduction in the percentage and number of these subsets within

Il17c
-/-
Apoe
-/-

mice versus

Apoe
-/-

controls. Explanted 12 week WD

Apoe
-/-

aortas treated with IL-17C resulted in the induction of multiple vascular chemokines and cytokines, and short-term homing experiments revealed diminished recruitment of Th17 cells to the aorta of

Il17c
-/-
Apoe
-/-

recipients. Smooth muscle cell-derived IL-17C plays a pro-atherogenic role by supporting the recruitment of Th17 cells to atherosclerotic lesions.
Abstract 588: Glyc-A, a Novel Inflammatory Biomarker, is Associated With Total and Non-Calcified Coronary Plaque Burden Beyond Traditional Cardiovascular Risk Factors
Authors: Joshi Aditya A , Lerman Joseph B , Aberra Tsion M , Kabbany Mohammad Tarek , Teague Heather L , Silverman Joanna I , Ng Qimin , Aridi Tarek Z , Baumer Yvonne , Salahuddin Taufiq S , Chen Marcus Y , Bluemke David A , Rodante Justin , Gelfand Joel M , Playford Martin P , Mehta Nehal N
Published: June 2021

Introduction:
Recent studies suggest that hsCRP may inaccurately predict coronary heart disease (CHD) in patients with chronic inflammatory disorders. GlycA, a novel inflammatory biomarker measured by nuclear magnetic resonance, was associated with future cardiovascular events in a large cohort study. Whether GlycA predicts CHD beyond hsCRP in chronic inflammatory patients is unknown. Psoriasis (PSO), a chronic inflammatory disease associated with increased cardiovascular risk, provides a clinical human model to assess the utility of GlycA as a risk marker of CHD.

Hypothesis:
We hypothesized that GlycA would associate with CHD, by total (TB) and non-calcified burden (NCB) assessed by coronary CT angiography, beyond traditional risk factors in PSO.

Methods:
151 consecutive PSO patients and 40 controls underwent coronary CT angiography (320 detector row) as part of a large cohort study (NCT01778569). Coronary plaque burden was assessed by QAngio (Medis). We measured GlycA by nuclear magnetic resonance (LabCorp). A physician ascertained clinical parameters. Labs were measured in a certified clinical research facility. Statistical analyses include multivariate regression and ROC modeling.

Results:
PSO patients were older, at low Framingham risk, but had significant cardiometabolic dysfunction and increased CHD by TB and NCB (Table). While hsCRP significantly associated with CHD in controls, it showed no relationship in PSO. GlycA, however, strongly associated with TB (β=0.14, p=0.01) and NCB (β=0.12, p=0.01) beyond traditional risk factors in PSO. Finally, ROC analyses demonstrated greater AUC for GlycA in predicting TB and NCB (Figure), suggesting GlycA adds incremental value to traditional cardiovascular risk factors.

Conclusion:
In conclusion, GlycA associates with coronary plaque burden in PSO. Our study suggests a role for GlycA beyond hsCRP in assessing CHD in inflammatory states. Larger prospective studies are needed to confirm these findings.
Abstract 593: Vascular Inflammation and Hypertension are Attenuated with T Cell Deletion of Serum and Glucocorticoid-regulated Kinase 1 (SGK1)
Authors: Norlander Allison E , Saleh Mohamed A , Pandey Arvind , Itani Hana A , Wu Jing , Dale Bethany , Harrison David G , Madhur Meena S
Published: June 2021

We have previously shown that the T cell-derived pro-inflammatory cytokine, interleukin 17A (IL-17A), is upregulated by and promotes angiotensin II-induced hypertension and contributes to vascular dysfunction. It was recently demonstrated that an excess of 40 mM of sodium chloride in culture enhances IL-17A production from CD4+ T cells in a Serum and Glucocorticoid-Regulated Kinase 1 (SGK1) dependent manner. We confirmed the effect of salt on CD4+ T cell differentiation and extended this finding to CD8+ T cells in which 40mM of excess salt increased the expression of IL-17A (4.7 fold, p=0.0003) and the salt-sensing kinase SGK1 (2.2 fold, p=.001) in naive CD8+ T cells cultured under Th17 polarizing conditions. Since dietary salt intake is associated with hypertension, we hypothesized that T cell SGK1 promotes hypertension and contributes to vascular dysfunction. To test this hypothesis, we crossed SGK1
fl/fl
mice with CD4cre mice to delete SGK1 in most T lymphocytes. Loss of T cell SGK1 resulted in a blunted blood pressure response to angiotensin II infusion (24.8 mmHg reduction, p=0.01) and DOCA-salt treatment (15.51 mmHg reduction, p<0.05). Moreover, vascular inflammation in response to angiotensin II infusion and/or DOCA-salt treatment was abrogated in these mice compared to SGK1
fl/fl
control mice. Angiotensin II increased total (CD45+) leukocytes in the aorta from 5.7 to 52.4x10
3
(p<0.01) in SGK1
fl/fl
mice compared to no increase in mice with T cell deletion of SGK1 (16.1 to 10.1x10
3
, p=ns). DOCA-salt induction increased total (CD45+) leukocytes in the aorta in SGK1
fl/fl
mice from 7.4 to 20.6x10
3
(p<0.05) compared to no increase in mice with T cell deletion of SGK1 (8.7 to 10.8x10
3
, p=ns). Furthermore, preliminary data show that angiotensin II infusion impairs vascular relaxation in mesenteric arteries isolated from SGK1
fl/fl
control mice, while this effect is blunted in angiotensin II treated mice with T cell deletion of SGK1. These studies demonstrate that T cell SGK1 may be a novel therapeutic target for hypertension and its associated vascular dysfunction.
Abstract 595: Blocking Cadherin-11 Adhesion After Myocardial Infarction Preserves Cardiac Function
Authors: Schroer Alison , Merryman W. David
Published: June 2021

Introduction:
Over one million Americans experience myocardial infarction (MI) every year, and the resulting scar and subsequent cardiac fibrosis contribute to heart failure and death. The cells primarily responsible for scar formation and cardiac fibrosis are cardiac fibroblasts (CFs), which differentiate into active myofibroblasts in response to injury, expressing a specialized adhesion protein: cadherin-11 (CDH11). CDH11 has recently been shown to contribute to inflammation and fibrosis in both rheumatoid arthritis and pulmonary fibrosis; therefore we hypothesized that blocking CDH11 adhesion after MI would reduce inflammation-driven infarct expansion and fibrotic remodeling to improve functional outcomes in mice.

Methods:
MI was induced in mice by ligation of a coronary artery, and mice were injected with a functional blocking antibody against CDH11 or a control IgG for 21 days. We assessed dynamic cardiac function with echocardiogram and measured changes in protein transcription and expression by qPCR and western blot.

Results and discussion:
Our preliminary results reveal an increase in both survival and cardiac function (ejection fraction) in the treated group relative to controls (A-B). Furthermore, increasing dilation of the left ventricle observed in the control was curtailed in the animals receiving the blocking antibody, resulting in significantly reduced total ventricle volume at 21 days post-MI (C-D). This reduced remodeling was preceded by reduced transcription of IL-6, a pro-inflammatory cytokine, in the antibody treated group three days post-MI (E). Our findings suggest that targeting CDH11-expressing myofibroblasts limits inflammation-driven remodeling while preserving cardiac function. The completion of this project will fully characterize phenotypic changes and tissue remodeling throughout the course of infarct healing, providing new biological insights, and highlighting a potential new treatment strategy for MI.
Abstract 599: Elucidating the Genetic Determinants of Extreme High-density Lipoprotein Phenotypes Using Next-generation Sequencing
Authors: Dron Jacqueline S , Wang Jian , McIntyre Adam D , Robinson John F , Ban Matthew R , Cao Henian , Rhainds David , Lettre Guillaume , Dubé Marie-Pierre , Tardif Jean-Claude , Hegele Robert A
Published: June 2021

HDL cholesterol (HDL-C) levels strongly associate with cardiovascular disease risk, and as a complex trait, are influenced by genetic and environmental factors. Extreme HDL-C concentrations are largely genetically determined; monogenic disorders of HDL-C have been well-characterized, including the primary candidate genes driving each extreme phenotype, which typically show autosomal recessive or co-dominant inheritance. Within genes causing syndromes of both HDL-C extremes, numerous disease-causing variants have been identified and functionally validated. In a unique cohort of patients with extreme HDL-C profiles (
N
=255), we applied our targeted next-generation sequencing panel LipidSeq
TM
, which is designed for clinical re-sequencing of genes associated with dyslipidemia and other metabolic disorders. We found that 20.6% and 11.8% of low (
N
=136) and high (
N
=119) HDL-C patients, respectively, carry heterozygous, large-effect mutations in pertinent genes explaining their phenotypes. To further characterize the genetic variation contributing to HDL-C levels, we next investigated the integrated polygenic contribution from multiple small-effect genetic variants using a polygenic trait score (PTS). We developed two scores to assess an individual’s burden of small-effect variants: one each for lowering and raising HDL-C levels. As a whole, low HDL-C patients had a significantly greater mean PTS for low HDL-C than normolipidemic controls (
P
<0.001); furthermore, there was no difference in PTS among carriers and non-carriers of large-effect variants. In contrast, high HDL-C patients’ mean PTS for high HDL-C in carriers of large-effect variants was not different from non-carrier or controls, while PTS in non-carriers was significantly greater than controls (both
P
<0.001). The findings confirm the complexity of extreme HDL-C levels and the differences in contributions of rare large-effect and common small-effect variants to these extremes.
Abstract 602: Development of Cell Therapy in Severe Familial Hypercholesterolemia
Authors: Koseki Masahiro , Saga Ayami , Kobayashi Takuya , Asaji Masumi , Okada Takeshi , Kawase Ryota , Tomita Koichi , Osuga Keigo , Ezoe Sachiko , Nishida Makoto , Sakata Yasushi , Yamashita Shizuya
Published: June 2021

Familial hypercholesterolemia (FH) patients are characterized by high LDL cholesterol levels in the blood and premature cardiovascular disease. Although most of heterozygous FH patients are able to be treated with statin, ezetimibe and bile acid sequestrants and anti-PCSK9 antibodies, homozygous FH patients are resistant to drug therapy. Therefore, many of homozygous FH patients used to be treated by LDL-apheresis. However, the effect of removing LDL cholesterol is still not sufficient and definitive therapies need to be developed.

With the increase of the evidence about the safety of mesenchymal stem cells and percutaneous transhepatic portal approach in islet transplantation, we have developed a cell transplantation therapy with allogeneic adipose tissue-derived multilineage progenitor cells, as an alternative treatment instead of liver transplantation.

We have generated a protocol for the phase 1 clinical study. The primary endpoint should be safety evaluation including allergic reaction, rejection reaction, thrombosis and complications in the blood vessel by cell-transplantation. The secondary endpoint should be the efficacy including changes in serum lipid such as LDL-C, TC, HDL-C, TG and Lp(a), and Secession possibility of LDL-apheresis.

We transplanted the first case in February, 2016. Then we are collecting clinical data. We would demonstrate the current status and the issues to be solved for the following study.
Abstract 608: Induction of Lysosomal Biogenesis in Adipose Tissue Macrophages Attenuates Inflammation and Cardiometabolic Disease
Authors: Evans Trent D , Sergin Ismail , Zhang Xiangyu , Bhattacharya Somashubhra , Dehestani Babak , Razani Babak
Published: June 2021

Adipose tissue inflammation is a fundamental feature of obesity and is strongly implicated in progression to overt cardiometabolic disease. In situ, this response is defined by a notable increase in number of adipose tissue macrophages (ATM’s) surrounding dying adipocytes and a phenotypic switch towards pro-inflammatory “M1” polarization. However, recent data suggest an equally notable upregulation of lysosomal catabolic pathways in these cells, and that inflammatory phenotypes may regulated by these processes. We and others speculate ATM lysosomal biogenesis is an adaptive response that can be harnessed for therapeutic benefit in treating cardiometabolic disease. To this end, we examined the effects of macrophage-specific overexpression of TFEB (mϕ-TFEB), a transcription factor master regulator of lysosomal biogenesis, in a murine model of diet induced obesity. We show broad physiological metabolic benefits including attenuated weight gain, improved body composition, and increased metabolic rate. Further, mϕ-TFEB mice demonstrate increased insulin sensitivity and glucose tolerance (Insulin and Glucose Tolerance Tests). These phenotypes are linked to profound changes in cellular, tissue, and systemic inflammation. mϕ-TFEB overexpression greatly reduces circulating interleukin 1β, and polarizes adipose tissue macrophages isolated from epididymal fat to an anti-inflammatory “M2” state in a manner that is dependent on lysosomal lipolysis. In cultured primary macrophages, TFEB overexpression attenuates LPS-induced proinflammatory M1 activation, and predisposes to IL-4 induced M2 polarization. Thus, harnessing the lysosomal biogenesis response in macrophages abrogates diet induced metabolic pathophysiology, potentially through regulation of inflammatory phenotypes. We postulate that ATM lysosomal biogenesis is a crucial, adaptive cellular response to obesity that can be preemptively induced to slow progression of cardiometabolic disease.
Abstract 612: Targeting I
κ
B Kinase β in Adipocyte Lineage Cells for Treatment of Obesity and Metabolic Dysfunctions
Authors: Helsley Robert N , Sui Yipeng , Park Se-Hyung , Liu Zun , Lee Richard G , Zhu Beibei , Kern Philip A , Zhou Changcheng
Published: June 2021

Objective:
Obesity is associated with a state of chronic low-grade inflammation that is a major contributor to diabetes and cardiovascular diseases. IκB kinase β (IKKβ), a central coordinator of inflammation through activation of NF-κB, has been identified as a potential therapeutic target for the treatment of obesity-associated metabolic dysfunctions. We recently revealed a novel role of IKKβ in adipogenesis and demonstrated that IKKβ functions in adipocyte precursor cells to regulate adipose tissue development. In this study, we used pharmacological and genetic approaches to explore the therapeutic potential of targeting IKKβ signaling in adipocyte lineage cells for treatment of obesity and metabolic dysfunctions.

Approach and Results:
We first evaluated an antisense oligonucleotide (ASO) inhibitor of IKKβ and found that IKKβ ASO ameliorated diet-induced metabolic dysfunctions in mice. Interestingly, IKKβ ASO also targeted IKKβ in adipocyte precursor cells, leading to inhibited adipocyte differentiation and reduced adiposity in high-fat (HF)-fed mice. Consistently, CRISPR/Cas9-mediated genomic deletion of IKKβ or pharmacological inhibition of IKKβ inhibited E3 ubiquitin ligase-mediated β-catenin ubiquitination and degradation in murine and human adipocyte precursor cells, resulting in elevated Wnt signaling and impaired adipocyte differentiation. To further elucidate the role of adipose progenitor IKKβ signaling in diet-induced obesity, we generated mice that selectively lack IKKβ in the white adipose lineage and confirmed the essential role of IKKβ in mediating adipocyte differentiation
in vivo
. Deficiency of IKKβ decreased HF-elicited adipogenesis in addition to reducing inflammation and protected mice from diet-induced obesity and insulin resistance.

Conclusions:
Our findings establish IKKβ as a pivotal regulator of adipogenesis and suggest that overnutrition-mediated IKKβ activation serves as an initial signal that triggers adipose progenitor cell differentiation in response to HF-feeding. Inhibition of IKKβ with antisense therapy may present as a novel therapeutic approach to combat obesity and metabolic dysfunctions.
Abstract 615: Association Between Multiple Modifiable Risk Factors of Cardiovascular Disease and Hypertension in a Rural Population of the United States
Authors: Mamudu Hadii M , Paul Timir , Wang Liang , Veeranki Sreenivas P , Panchal Hemang B , Alamian Arsham , Subedi Pooja , Budoff Matthew
Published: June 2021

Background:
Hypertension (HTN) is one of the major risk factors for cardiovascular diseases (CVD) that afflicts one-third of the population in United States (US). This study examined the association between multiple modifiable risk factors for HTN in a rural hard-to-reach population.

Methods:
During January 2011 and December 2012, 1629 community-dwelling asymptomatic individuals from central Appalachia participated in screening for subclinical atherosclerosis, during which the participants were asked to report whether a physician or health worker has informed them that they had HTN (yes/no). Additionally, baseline data consisting of two non-modifiable risk factors (sex, age) and 5 modifiable risk factors (obesity, diabetes, hypercholesterolemia, smoking, and sedentary lifestyle) were collected. Descriptive statistics involving prevalence of risk factors and multivariate logistic regression analyses to determine the strength of association between hypertension and the number of risk factors were conducted.

Results:
Of the 1629 study participants, about half (49.8%) had hypertension. Among hypertensive patients, 31.4% were obese and 62.3% having hypercholesterolemia. Overall, having 2 risk factors consisted the largest group of participants with HTN. After adjusting for the non-modifiable risk factors (sex, age), obesity and diabetes increased the odds of having HTN by more than two folds ([OR=2.02, CI=1.57-2.60] and [OR=2.30, CI=1.66-3.18], respectively) and hypercholesterolemia and sedentary lifestyle increased the odds for HTN by more than one fold ([OR=1.26, CI=1.02-1.56) and [OR=1.38, CI=1.12-1.70], respectively). Compared to those without HTN, having 2, 3, and 4 or 5 modifiable risk factors were significantly associated with increased odds of having HTN by about two-folds [OR=1.72, CI=1.21-2.44], two and half folds [OR=2.55, 1.74-3.74], and six folds [OR=5.96, 3.42-10.41], respectively.

Conclusion:
The study suggests that odds of having HTN increases with the number of modifiable risk factors for CVD. Hence, by implementing an integrated CVD program for treating and controlling modifiable risk factors of HTN would decrease the future risk of CVD and help to achieve the
2020 Impact Goal
of the American Health Association.