bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 470: Ultrasound Carotid Plaque Features, Cardiovascular Disease Risk Factors and Events: the Multi-Ethnic Study of Atherosclerosis
Authors: Mitchell Carol , Korcarz Claudia E , Gepner Adam D , Kaufman Joel D , Post Wendy S , Tracy Russell , Gassett Amanda J , Ma Nanxun , McClelland Robyn L , Stein James H
Published: March 2019

Background:
Grayscale ultrasound atherosclerotic plaque characteristics may predict cardiovascular disease (CVD) events, though they have not been investigated in a large primary prevention cohort. This study determined if CVD risk factors were associated with carotid plaque ultrasound characteristics and if these characteristics could predict future coronary heart disease (CHD) and stroke/transient ischemic attack (TIA) events in a multi-ethnic cohort free of known CVD at baseline.

Methods:
We measured carotid artery total plaque area (TPA) and grayscale carotid plaque features (grayscale median, black areas, and discrete white areas) at baseline in participants of the Multi-Ethnic Study of Atherosclerosis that had B-mode carotid ultrasound examinations. There were 2205 participants with images available for TPA analyses and 1703 with images available for grayscale analyses. Multivariable linear regression analysis was used to examine relationships between baseline ultrasound carotid plaque features and CVD risk factors. Cox proportional hazards models were used to assess their ability to predict incident CHD and stroke/TIA events over an average follow-up of 13.3 years. The predictive characteristics of TPA and carotid plaque features were compared to carotid plaque score and coronary artery calcification (CAC) score.

Results:
Participants were mean (standard deviation) 65.4 (9.6) years old, 49% male, 39% White, 28% Black, 22% Hispanic, and 11% Chinese. Mean TPA (27.7 [24.7] mm
2
), but not grayscale plaque features, were associated with several CVD risk factors. In risk factor-adjusted models, TPA was the only plaque feature that predicted incident CHD events (HR 1.23; 95% CI 1.11-1.36; p<0.001) with C-statistics for CHD that were similar to carotid plaque score, but lower than CAC score. TPA did not independently predict stroke/TIA events. No gray scale plaque feature predicted future CHD or stroke/TIA events.

Conclusions:
In middle-aged individuals free of known CVD, carotid TPA was associated with CVD risk factors and predicted incident CHD events while grayscale plaque features did not. For CHD, predictive characteristics of TPA were similar to carotid plaque score but lower than CAC score.
Abstract 476: The Role of N-glycosylation in the Myogenic Differentiation of Stem Cells to Vascular Smooth Muscle Cells
Authors: Corcoran Eoin , Olayinka Abidemi , O'Connor Brendan , Cahill Paul A
Published: March 2019

Cell fate decisions within the vasculature are crucial to the pathogenesis of vascular diseases, including, arteriosclerosis, atherosclerosis and restenosis after angioplasty. Notch signalling is involved in regulating cell fate in vasculature development during embryogenesis resulting in altered cell fate decisions leading to vascular disease. The Notch signalling pathway is highly regulated by a number of mechanisms including glycosylation, a post-translational modification.

Our main objective was to define a putative role for N-glycosylation of the Notch1 receptor in controlling resident vascular stem cell fate
in vitro
. Utilising ligand Jagged-induced Notch signalling assay, qRT-PCR, immunocytochemistry, ectopic expression of Notch1 receptor, siRNA knockdown, pharmacological inhibition and enzyme linked lectin assay (ELLA), alterations in N-glycan decoration of the Notch1 receptor were assessed before evaluation of their effects on Notch signalling and Notch ligand promotion of myogenic differentiation.

N-glycosylation of the Notch1 receptor was assessed using a combination of the HPLC and ELLA assays and confirmed the presence of N-glycans on the receptor, an effect that was abrogated following inhibition of glycosyltransferase activity with tunicamycin and lunatic fringe (Lfng) knockdown. Jagged1-induced Notch activation increased Notch target gene expression and promoted myogenic differentiation of bone-marrow derived mesenchymal stem cells and resident vascular stem cells. Selective knockdown of the Notch1 receptor in stem cells resulted in a significant decrease in Jagged1 stimulated Hey1 expression, a Notch1 target gene, concomitant with a reduction in myogenic differentiation due to decreased smooth muscle differentiation marker expression (CNN1 and MYH11 mRNA and protein levels). Inhibition of N-glycosylation with tunicamycin lead to a down regulation of smooth muscle differentiation markers, CNN1 and MYH11 independent of a reduction in Notch target gene expression. Lfng knockdown lead to a similar significant reduction in Jagged1 induced myogenic differentiation (reduced CNN1 expression). Collectively, these results suggest that N-glycosylation of Notch1 receptor is involved in Notch signalling leading to altered resident vascular stem cell fate.
Abstract 477: Platelet microRNAs Crosstalk with Vascular Endothelial Cells in Diabetes
Authors: Dangwal Seema , Stratmann Bernd , Ke Xiao , Kleinbongard Petra , Heusch Gerd , Erbel Raimund , Tschoepe Diethelm , Thum Thomas
Published: March 2019

Platelets are major source of extracellular miRNAs (miRs) constituting the plasma miR-pool. Here we generated miR-profile of leukocyte depleted platelets (LDP) isolated from type-2 diabetic (T2DM) or healthy subjects and investigated mechanism of miRNA mediated vascular crosstalk.

LDPs were obtained by leukocyte filtration of platelet rich plasma isolated from blood withdrawn from T2DM (6 males, age: 54.8±7.6 yr; HbA1c: 8.9±0.6%; BMI: 30.9±2.8kg/m2) and healthy subjects (10 males, age: 49.2±6.8yr; HbA1c: 5.3±0.2%; BMI: 26.6±3.8kg/m2). MiR-profile was generated using total RNA isolated from T2DM or healthy LDPs. Students t-test applied to compare groups and p≤0.05 considered significant.

Among all detected 735 miRs (cutoff<29 Ct), 19.5% miRs were depleted, whereas 7.5% showed >2 fold increase in T2DM-LDPs. Compared to healthy LDPs (n=10), depletion of 14 platelet-enriched miR candidates, including miR-21 and -22, were validated by RTPCR in individual T2DM-LDPs (n=11) irrespective of aspirin intake or presence of coronary heart disease.Ex-vivo thrombin stimulation of washed healthy platelets increased the release of miRNA into cellular-supernatants and confocal microscopy confirmed the cellular uptake of platelet released miRNA-carrying microparticles into human coronary artery endothelial cells (ECs) within 2-24h. In addition, EC overexpression of miR-21 or -22 decreased cell proliferation, whereas miR-22 also attenuated their tube forming capacity. MiR-21 or -22 modulations inversely regulated gene expression of their common predicted target, the cell-cycle regulator gene- CDK-6. MiR concentrations were also measured in particulate debris and plasma derived from coronary aspirates retrieved during stent implantation from 3 T2DM or 4 non-diabetic patients. In line, higher miR-21 or -22 ratios in plasma to particulate debris in T2DM vs non-diabetic aspirates indicated miR release from particulate debris into coronary plasma. This in turn provides relatively richer miR-21 or -22 microenvironment to the vicinal vascular ECs.

T2DM platelets show distinct miR-profile compared to healthy platelets and depletion of miRNAs may represent their active secretion from diabetic platelets. Altered coronary microenvironment may lead to altered cellular responses to injury in diabetic vessels.
Abstract 485: Novel Mechanisms at Immune-vascular Interfaces Regulates Myogenic Tone of Resistance Arteries and Induce Hypertension in Mice
Authors: Carnevale Daniela , Vinciguerra Iolanda , Casaburo Manuel , Perrotta Marialuisa , Iacobucci Roberta , Iodice Daniele , Lembo Giuseppe
Published: March 2019

In the search for mechanisms regulating the pathophysiological relationship existing between hypertension and immune system, CD8 effector T cells emerge as possible mediators of target organ colonization. However, how the crosstalk at vascular interfaces is established still remains unidentified. Phosphatidylinositol-3-kinase gamma (PI3Kγ) is an intracellular signaling involved in the acquisition of CD8 effectors functions and was previously showed by our group as involved in hypertension. The hypothesis here tested rely on the possibility that PI3Kγ could regulate trafficking of immune cells at vascular interfaces relevant for hypertension. By taking advantage of a knock-in mouse model, expressing a constitutively active PI3Kγ isoform (PI3Kγ
CX/CX
), we conceived an experimental setting allowing to assess the direct crosstalk between immune cells and vasculature, obtained by co-culturing CD8 T cells with isolated resistance arteries mounted in pressurized systems. Thus, CD8 isolated from PI3Kγ
CX/CX
mice were co-cultured for 3 days with WT resistance arteries and, after the incubation period, vascular function was tested by challenging arteries to increasing intraluminal pressure to detect myogenic tone (MT). CD8 T cells with constitutively active PI3Kγ spontaneously increased MT of mesenteric arteries, a behavior typically associated with hypertension. Conversely, the same T cell population from WT mice, had no effect when cultured with vessels. In addition, PI3Kγ
CX/CX
CD8 infiltrated naïve vessels upon co-culture suggesting an intrinsic ability to colonize resistance districts relevant for hypertension. To test the in vivo relevance of the observed phenotype, we performed an adoptive transfer of PI3Kγ
CX/CX
CD8 in WT mice, finding that they developed spontaneous hypertension, accompanied by infiltration of effector CD8 in perivascular spaces of resistance districts as kidneys. Taken together these data show that PI3Kγ signaling in CD8 T cells is crucial for their effector functions in target vasculature of hypertension, likely contributing to BP increase by increasing peripheral resistance. These results also suggest translational potential as PI3Kγ inhibitors could modulate the immune response involved in hypertension.
Abstract 493: Neutrophil and Macrophage Cell Surface Colony-Stimulating Factor-1 is Shed by a Disintegrin and Metalloprotease 17 and Contributes to Stimulating Macrophage Proliferation in Inflammation
Authors: Tang Jingjing , Frey Jeremy M , Wilson Carole L , Moncada-Pazos Angela , Levet Clemence , Freeman Matthew , Stanley E. R , Rosenfeld Michael E , Bornfeldt Karin E , Raines Elaine W
Published: March 2019

Recent studies highlight a role for macrophage proliferation post monocyte recruitment in inflammatory conditions, such as atherosclerosis. However, the mechanisms regulating macrophage proliferation are not well understood. Using an acute peritonitis model, we identified a 40 ± 8% reduction (mean ± SEM; n=5; p<0.01) in macrophages in the S phase of the cell cycle 40 hrs post thioglycollate injection in mice lacking the transmembrane protease ADAM17 in hematopoietic cells. ADAM17 is a member of a disintegrin and metalloprotease family that cleaves many cell surface proteins involved in inflammation. The macrophage proliferation defect in ADAM17 null chimeras was associated with a 60 ± 10% (n=5; p<0.001) decrease in soluble macrophage colony-stimulating factor (CSF)-1 in the peritoneum, and was rescued by intraperitoneal injection of CSF-1. We demonstrate that neutrophils, the first innate immune cell that migrates into inflammatory sites, and macrophages are both major sources of cell surface (cs)CSF-1 in this acute inflammation model, adding to the current knowledge that stromal, epithelial and endothelial cells are sources of CSF-1. We show that ADAM17 is critical in mediating csCSF-1 release in the inflamed peritoneum, and that this release following neutrophil extravasation is associated with elevated expression of iRhom2, a member of the rhomboid-like superfamily, which promotes ADAM17 maturation and trafficking to the cell surface. Accordingly, deletion of hematopoietic iRhom2 is sufficient to prevent csCSF-1 release from neutrophils and macrophages, thus recapitulating the defect in macrophage proliferation seen in ADAM17 null cells. Our data indicate that csCSF-1 release and macrophage proliferation is self-limiting in states of acute inflammation due to the transient nature of leukocyte recruitment and temporally restricted csCSF-1 expression and release. In chronic inflammation such as atherosclerosis, we show that macrophage proliferation is decreased by 30 ± 7% (n=6; p<0.01) in atherosclerotic lesions of ADAM17 hematopoietic null LDL receptor-deficient mice fed a high fat diet for 16 weeks. Together, these results demonstrate a novel mechanism governing macrophage proliferation in both acute and chronic inflammation.
Abstract 494: Endothelial Cell Heterogeneity Governs Basal and Inducible Inflammatory Responses Relevant to Allograft Injury
Authors: Zufall Gianna , Valenzuela Nicole
Published: March 2019

Introduction:
Transplanted organs are not equally susceptible to rejection, suggesting a role for site specialization of vascular endothelial cells (EC). Most studies of leukocyte recruitment employ umbilical vein or aorta EC, and fail to capture physiological differences among EC.

Methods:
EC were left unstimulated or treated with TNFα or IL-1β (0.01-10ng/mL, 4-24hr). Confluent primary human EC from aorta (AEC), coronary artery (CAEC), cardiac microvessel (CMVEC), pulmonary artery (PAEC), lung microvessel (LMVEC), skin blood microvessel (DMVEC), liver sinusoids (LSEC), kidney glomerulus (RGEC) and brain microvessel (HMBVEC) were compared for expression of adhesion molecules and chemokines by Nanostring Human Immunology 594 gene panel, flow cytometry and 38-plex Luminex assay (n=3 donors). A novel adhesion assay enabled immunophenotyping of allogeneic leukocyte subsets (T, B, NK and monocytes) adherent to EC (n=4 PBMC donors).

Results:
>200 immunology-related genes were differentially expressed (fold >1.5, p<0.05) across untreated EC. Microvascular EC expressed higher baseline levels of ICAM-1, E-selectin and VCAM-1. TNFα and IL-1β increased these adhesion molecules, but there were clear differences in the magnitude and kinetics across cells. Most notably, LSEC failed to significantly upregulate E-selectin, and exhibited the lowest adherence of leukocytes. Only LSEC constitutively expressed suppressors of cytokine signaling (SOCS1 and SOCS3).

After stimulation with TNFα and IL-1β, cardiac EC produced more fractalkine than other EC irrespective of vessel size; but large vessel (AEC, PAEC) produced the most IL-6 and G-CSF. AEC were a poor source of RANTES and IP-10, while LSEC did not increase fractalkine or GM-CSF.

IL-1β increased GM-CSF, G-CSF, and GROα and IL-6. TNFα increased RANTES. More NK and B cells were recruited to TNFα than IL-1β activated EC. IL-1β selectively increased the nuclear cofactor NFKBIZ (IκBζ), but TNFα promoted Bcl-3 expression, suggesting different transcriptional programs.

Conclusion:
These findings demonstrate heterogeneity among EC from different vascular beds and organs. SOCS proteins may be potential therapeutic targets to reduce the inflammatory responses of ECs in multiple vascular disease models.
Abstract 495: Nicotine Promotes Endothelial Dysfunction and Vascular Inflammation in Diet-induced Obese Rats: Role of Macrophage TNFa
Authors: Liu Chang , Wang Aimei , Hao Yun , Huang Lei , Zhihang Yang , Tian Runxia , Raij Leopoldo , Zhou Ming Sheng
Published: March 2019

Obesity and cigarette smoke are major cardiovascular (CV) risk factors and, when coexisting in the same individuals, have additive/synergistic effects upon CVD. We studied the mechanisms involved in nicotine enhancement of CVD in Sprague Dawley rats with diet-induced obesity
.
The rats were fed either a high fat (HFD) or normal rat chow (NFD) diet with or without nicotine (100 mg/L in drinking water) for 20 weeks. HFD rats developed central obesity, increased systolic blood pressure (SBP 146 ± 3 vs. 130 ± 5 mmHg in NFD , p<0.05), aortic superoxide (O
2
-
1357 ± 142 vs. 847 ± 102 counts/min/mg in NFD, p<0.05) production, and impaired endothelial nitric oxide synthase (eNOS) and endothelium-dependent relaxation to acetylcholine (EDR, Emax: 85 ± 5 vs. 99 ± 1% in NFD, p<0.05). Nicotine further increased SBP (159 ± 5 mmHg, p<0.05), O
2
-
(1689 ± 87 counts/min/mg, p<0.05) and impaired eNOS and EDR (Emax: 72 ± 4%, p<0.05) in obese rats. In the peritoneal macrophages from obese rats, tumor necrosis factor (TNF) α, interleukin 1β and CD36 were increased, and were further increased in nicotine-treated obese rats. Using PCR array we found that 3 of 84 target proinflammatory genes were increased by 2-4 fold in the aorta of obese rats, 11 of the target genes were further increased in nicotine-treated obese rats. HUVECs, incubated with conditioned medium from the peritoneal macrophages of nicotine treated-obese rats, exhibited reduced eNOS and increased NADPH oxidase subunits gp91phox and p22phox expression. Those effects were partially prevented by adding anti-TNFα antibody to the conditioned medium. Our results suggest that nicotine aggravates the CV effects of diet–induced obesity including the oxidative stress, vascular inflammation and endothelial dysfunction. The underlying mechanisms may involve in targeting endothelium by enhancement of macrophage-derived TNFα.
Abstract 501: Angiotensin II Increases Angiogenesis by Nf-κB-mediated Transcriptional Activation of Angiogenic Factor Aggf1
Authors: Si Wenxia , Xie Wen , Deng Wenbing , Karnik Sadashiva S , Xu Chengqi , Chen Qiuyun , Wang Qing K
Published: March 2019

Angiogenic factor AGGF1 is involved in the specification of hemangioblasts, vascular development, differentiation of veins, angiogenesis, neointimal formation after vascular injury, and cardiac hypertrophy and heart failure. However, the regulation of the expression of AGGF1 remains incompletely characterized. In this study, we show that vasoconstrictor angiotensin II (AngII), the major effector of the renin-angiotensin system (RAS) and one of the most important regulators of the cardiovascular system, induces the expression of AGGF1 through NF-κB, and up-regulation of AGGF1 plays a key role in AngII-induced angiogenesis. AngII significantly up-regulated the levels of AGGF1 mRNA and protein at low doses (10-40 μM), but not at high doses in HUVECs (>60 μM). AngII receptor AT1R inhibitor losartan inhibited AngII-induced up-regulation of AGGF1, whereas AT2R inhibitor PD123319 further increased AngII-induced up-regulation of AGGF1, suggesting that up-regulation of AGGF1 by AngII was through AT1R. Up-regulation of AGGF1 by AngII was blocked by NF-κB inhibitors PDTC and quinacrine. A binding site for p65 was identified at the 5’-UTR of
AGGF1
and shown to bind p65 directly using chromatin immunoprecipitation analysis (CHIP) and electrophoretic mobility shift assays (EMSA). Western blot, real-time RT-PCR and luciferase assays revealed that p65 transcriptionally activated
AGGF1
expression. Capillary tube formation assays showed that knockdown of
AGGF1
expression inhibited angiogenesis induced by AngII. These data suggest that AngII acts as a critical regulator of AGGF1 expression through NF-κB, and AGGF1 plays a key role in AngII-induced angiogenesis.
Abstract 502: Glucose-6-Phosphate Dehydrogenase Regulates MYH11 and MYOCD Expression in HDAC and miR-1 Dependent Manner
Authors: Dhagia Vidhi , Joshi SachindraRaj , Soldatos Vasiliki , Edwards John G , Gupte Sachin A
Published: March 2019

Vascular smooth muscle cell (VSMC) phenotype and function is altered in diabetic and obese patients and animals. Increased activity of glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme in Pentose Phosphate Pathway, in diabetic and obese animals is associated with decreased expression of MYOCD and MYOCD-dependent genes in the arteries of these animals. Therefore, we hypothesize that reprogrammed metabolism and increased G6PD activity is critical in switching the VSMC from a differentiated to a dedifferentiated phenotype. Our results demonstrated metabolic reprogramming and decrease in MYOCD and MYOCD-driven MYH11 in the aorta of normal chow- and high fat diet (HFD)-fed diabetic Goto-Kakizaki (GK) as compared to control rats. Up regulated G6PD activity in the aorta of these animals positively correlated with increased arterial elastance (R
2
= 0.8322) and total peripheral resistance (R
2
= 0.8364).
In vitro
, VSMCs (A7r5) treated with Ad-shRNA for G6PD, epiandrosterone (Epi), G6PD inhibitor, and PD2958, a selective inhibitor of G6PD activity, increased (P<0.05)
Myocd
and
Myh11
levels and MYOCD-regulated MYH11 and CNN1 expressions as compared to untreated cells. Furthermore, we found that G6PD-mediated expression of MYOCD and MYH11 expressions was regulated at transcriptional and post transcriptional level. Inhibition of G6PD by PD2958 or Epi decreased histone deacetylase (HDAC) activity by 70-80% (P<0.05), and concomitantly, PD2958 and Ad-shRNA-G6PD up regulated H3K9Ac expression in A7r5 cells as compared to their respective controls. Next, we compared the expression of VSMC-restricted MYH11 in A7r5 cells treated with HDAC inhibitor, Trichostatin (TSA), and PD2958+TSA. Our results demonstrated that MYH11 expression increased by TSA and PD2958 was not further potentiated by PD2958+TSA, suggesting that G6PD inhibition up regulated MYH11 via HDAC inhibition. We also found that G6PD inhibition increased miR-1 levels, which post-transcriptionally regulates MYOCD and MYOCD-dependent gene expression. Therefore, our results collectively suggest that increased G6PD activity is associated with switching of VSMCs from a differentiated to a dedifferentiated phenotype in HDAC and miR-1 dependent manner.
Abstract 503: Smooth Muscle Cell Cytochrome B5 reductase < CYB5R3 > expression modulates vascular tone
Authors: Durgin Brittany G , Hahn Scott A , Miller Megan P , Miller Megan P , Straub Adam C
Published: March 2019

Hypertension is a major risk factor for cardiovascular-related morbidity and mortality. Nitric oxide (NO) resistance results in an inability of arterial blood vessels to relax contributing to hypertension. NO relaxes vascular smooth muscle cells (SMCs) by stimulating soluble guanylyl cyclase (sGC) to generate cyclic guanosine monophosphate (cGMP) which leads to downstream activation of protein kinase G (PKG) and vasodilation. Importantly, the sGC heme iron is required to be in the reduced (Fe
2+
) state in order for NO to bind and activate sGC. When the sGC heme iron is oxidized (Fe
3+
), sGC is insensitive to NO signaling for cGMP production, resulting in hypertension. We recently published evidence that NADH cytochrome b5 reductase 3 (CYB5R3) is a sGC heme reductase that maintains sGC in the reduced (Fe
2+
), NO-sensitive state. Transient knockdown and pharmacological inhibition of CYB5R3 in SMCs blocks NO-mediated cGMP production and aortic relaxation. Combined, these data implicated CYB5R3 as a potential regulator of systemic blood pressure. To test this, we created tamoxifen-inducible SMC-specific CYB5R3 knockout mice (SMC CYB5R3 KO) and measured systemic blood pressure. We found SMC CYB5R3 KO mice have ~ 5 mmHg increase in mean arterial blood pressure at baseline relative to SMC CYB5R3 WT mice. Subsequently, Angiotensin II (Ang II) infusion resulted in a further ~ 15 mmHg increase in blood pressure respective to SMC CYB5R3 WT mice. Future studies will test whether SMC CYB5R3 may confer vascular protection against Ang II-induced HTN, vessel fibrosis, and SMC dysfunction by maintaining sGC in the reduced, NO sensitive state
.
Abstract 506: Pharmacological Inhibition of Forkhead-box Class O Transcription Factors Reduces Nitric Oxide Signaling Through Downregulation of Soluble Guanylate Cyclase
Authors: Galley Joseph C
Published: March 2019

Dysregulated vascular tone contributes to hypertension. Nitric oxide (NO) activated soluble guanylate cyclase (sGC) is an integral player in the regulation of vasodilation capacity through production of the secondary messenger cyclic guanosine monophosphate (cGMP) to induce vasorelaxation. Impaired NO-stimulated cGMP production through sGC has previously been shown to significantly contribute to the development and acceleration of hypertension. To date, little is understood concerning the transcriptional regulation of sGC expression in vascular smooth muscle cells (VSMCs). It has been established that the class O forkhead-box (FoxO) family of transcription factors play important roles in the regulation of cell growth, proliferation, metabolism, survival, lifespan, and tissue differentiation. Therefore, we hypothesize that FoxO transcription factors play an important role in sGC regulation and hypertension. Here we show that pan-pharmacological inhibition of FoxO transcription factors using AS1842856 significantly reduced sGC mRNA expression rapidly and in a dose dependent manner. Similarly, sGC protein expression significantly decreased quickly and dose-dependently upon FoxO protein inhibition. Furthermore, VSMC FoxO inhibitor pre-treatment significantly decreased cGMP production under basal and after stimulation with DEA-NONOate, a NO-donor molecule. Functionally,
Ex vivo
two-pin myography experiments show that FoxO inhibitor-treated C57BL/6 mouse aortas have significantly blunted sodium nitroprusside (SNP)-induced (NO-dependent) vasorelaxation. Taken together, our data suggest FoxO transcription factor activity is necessary for the maintenance of sGC expression and NO-dependent vascular tone modulation.
Abstract 508: Endothelial Cell Telomerase Promotes Aberrant Cell Proliferation in Pulmonary Hypertension
Authors: Hyzny Eric , Doyon Genevieve , Goncharova Elena , Bruemmer Dennis
Published: March 2019

Background:
Pulmonary arterial hypertension (PAH) is a progressive, severe disease with high morbidity and mortality. Mechanistically, early pulmonary vasoconstriction promotes cell proliferation and extensive vascular remodeling. Mammalian cell proliferation is dependent on telomerase expression; however, a mechanistic link between endothelial cell telomerase reverse transcriptase (TERT) and the control of mitogenic responses during the development of PAH has never been established.

Hypothesis:
Elevated telomerase levels in the diseased pulmonary endothelium induce proliferation of endothelial and smooth muscle cells in the pulmonary vasculature.

Methods:
We first received isolated pulmonary arterial endothelial cells from control subjects and patients with PAH. Autopsy sections obtained from patients were stained for endothelial cells, mitogenic markers, and TERT. Pooled primary pulmonary endothelial cells were used for in vitro experiments.

Results:
TERT expression was increased in the endothelium of pulmonary artery specimens of patients with PAH. Co-staining for the mitogenic marker PCNA confirmed high TERT expression specifically in proliferating cells. Key mechanistic stimuli causally involved in PAH, including hypoxia, inflammation, and vascular injury, increased mRNA and protein expression of TERT in pulmonary artery endothelial cells. TERT expression is necessary and sufficient for endothelial cell proliferation, as demonstrated through genetic TERT depletion and overexpression. Mechanistically, TERT supports the transcription of key endothelial growth factors, including vascular endothelial growth factor and epidermal growth factor, an effect that is mediated through TERT-dependent regulation of the transcription factor HIF1α.

Conclusions:
These data support the hypothesis that TERT promotes a proliferative phenotype of endothelial cells in PAH. Future experiments will investigate the cell-specific role of TERT in endothelial cell function and proliferation during the development of PAH using Cre/loxP recombination in mice harboring floxed TERT alleles.
Abstract 509: Fibronectin Containing Extra Domain a Modulates TLR4-Dependent Smooth Muscle Cell Proliferation and Neointimal Hyperplasia in Apolipoprotein E-Deficient Mice
Authors: Jain Manish , Doddapattar Prakash , Chorawala Mehul , Dhanesha Nirav , Chauhan Anil
Published: March 2019

Background:
Fibronectin-splice variant containing extra domain A (Fn-EDA), an endogenous ligand of toll-like-receptor 4 (TLR4), is a characteristic feature of proliferating vascular smooth muscle cells (VSMCs) that contributes to intimal thickening and atherosclerosis. Very little is known about the role of Fn-EDA in VSMC proliferation or its clinical implication in neointima formation after vascular injury in the comorbid condition of hyperlipidemia.

Methods:
To explore the mechanism and therapeutic potential of targeting Fn-EDA in vascular diseases, we used carotid wire-injury model to induce neointimal hyperplasia (28 days) in hyperlipidemic Apoe
-/-
, Fn-EDA
-/-
Apoe
-/-
, TLR4
-/-
Apoe
-/-
, Fn-EDA
-/-
TLR4
-/-
Apoe
-/-
and SMC- specific Fn-EDA
-/-
mice (Fn-EDA
fl/fl
αSMACre
+
Apoe
-/-
) (male and female; 10 weeks old). Furthermore, VSMCs were isolated from different genotypes and stimulated with PDGF to dissect the mechanisms.

Results:
Using immunostaining, we found increased expression of Fn-EDA in human atheroma, injured carotid artery and proliferating VSMCs of Apoe
-/-
mice (P<0.05 vs. control). Irrespective of gender, genetic deletion of Fn-EDA in Apoe
-/-
mice reduced neointimal area after vascular injury (P<0.05 vs. Apoe
-/-
). Aortic VSMCs isolated from Fn-EDA
-/-
Apoe
-/-
mice exhibited reduced cell proliferation (BrdU assay, G1/S phase transition) and migration (wound healing assay) that was concomitant with elevated pAKT2 and pFOXO4 expression and reduction in pAKT1, pmTOR, pNFκB, TNF-α and IL-1β secretion levels (P<0.05 vs. Apoe
-/-
). Genetic ablation of TLR4 in Apoe
-/-
mice reduced neointimal hyperplasia concomitant with decreased VSMCs proliferation and migration (P<0.05 vs. Apoe
-/-
), but had no additive effect in Fn-EDA
-/-
Apoe
-/-
mice. Purified cellular Fn, which contains EDA, attenuated pAKT2 and pFOXO4 and potentiated pAKT1, pmTOR, pNFκB, TNF-α and IL-1β secretion in SMCs from Fn-EDA
-/-
Apoe
-/-
mice (P<0.05 vs. plasma Fn that lacks Fn-EDA) but not from Fn-EDA
-/-
TLR4
-/-
Apoe
-/-
mice. Finally, SMC-specific Fn-EDA
-/-
mice (Fn-EDA
fl/fl
αSMACre
+
Apoe
-/-
) exhibited reduced neointimal hyperplasia.

Conclusion:
Fn-EDA modulates TLR4-dependent VSMCs proliferation and intimal hyperplasia after vascular injury in the comorbid condition of hyperlipidemia.
Abstract 513: ACE2-EPCs Protect Ecs Majorly Through Their Exosomal Effects on Mitochondria
Authors: Wang Jinju , Ma Xiaotang , Cheng Chuanfang , Chen Shuzhen , Liu Shiming , Zhao Bin , Bihl Ji
Published: March 2019

Angiotensin-converting enzyme 2 (ACE2) is an emerging cardiovascular protective target which mediates the metabolism of angiotensin (Ang) II into Ang (1-7). Our group has demonstrated that ACE2 overexpression enhances the therapeutic efficacy of endothelial progenitor cells (EPCs) for ischemic stroke. Here, we investigated whether ACE2-primed EPCs (ACE2-EPCs) can protect cerebral microvascular endothelial cells (ECs) against injury and dysfunction in an
in vitro
model, with focusing on their exosomal and cytokine paracrine effects on endothelial mitochondria. Human EPCs were transfected with lentivirus containing null or human ACE2 cDNA (denoted as Null-EPCs, ACE2-EPCs, respectively). Their conditioned culture media, w/wo ultra-centrifugation for depletion of exosomes (ACE2-EPC-CM
EX-
, Null-EPC-CM
EX-
, ACE2-EPC-CM and Null-EPC-CM), were used for co-culture experiments. ECs injury and dysfunction model was induced by Ang II (10
-7
M) for 8 hrs before 16 hr co-culture. After that, ECs were collected for analyses of apoptosis, angiogenic ability, mitochondrion functions (ROS production, membrane potential, fragmentation), and gene expressions (ACE2, Nox2 and Nox4). The co-culture medium was collected for measuring the levels of ACE2, Ang II / Ang-(1-7) and growth factors (VEGF and IGF). Our results showed that: 1) ACE2-EPC-CM had higher levels of ACE2, Ang (1-7), VEGF and IGF than that of Null-EPC-CM. 2) Ang II-injured ECs displayed increase of apoptotic rate and reduction in tube formation and migration abilities, which were associated with ACE2 downregulation, Ang II / Ang (1-7) imbalance, Nox2/Nox4 upregulation, ROS overproduction, increase of mitochondrion fragmentation and decrease of membrane potential. 3) ACE2-EPC-CM had better protective effects than Null-EPC-CM on Ang II-injured ECs, which were associated with the improvements on ACE2 expression, Ang II / Ang (1-7) balance and mitochondrial functions. 4) ACE2-EPC-CM
EX-
and Null-EPC-CM
EX-
had remarkably reduced effects by over 70% as respectively compared to ACE2-EPCs-CM and Null-EPCs-CM. In conclusion, our data demonstrate that ACE2 overexpression can enhance the protective effects of EPCs on ECs injury, majorly through the exosomal effects on mitochondrial function.
Abstract 516: Endothelial Foxp1 Regulates Cardiac Remodeling via Tgfbeta and Et-1 Signal Pathway
Authors: Liu Jie , Zhuang Tao , Chen Xiaoli , Pi Jinjiang , Kuang Yashu , Peng Sheng , Zhang Lin , Zhang Yuzhen
Published: March 2019

Rationale:
Cardiac fibrosis and hypertrophy is common features in left ventricular remodeling that leads to heart failure. Forkhead box protein P-1 (Foxp1), a large modular transcriptional repressor that binds to DNA via the highly conserved forkhead DNA-binding domain, is crucial in coordinating the balance of cardiomyocyte proliferation and differentiation through cell lineage-specific regulation during cardiac development. However, its role in adult pathological cardiac remodel remains to be clarified.

Objective:
We seek to determine the role and the underlying mechanisms of Foxp1 in cardiac fibrosis and hypertrophy.

Methods and Results:
Foxp1 was highly expressed in endocardium and cardiac microvascular endothelial cells, significantly reduced following AngII stimulation. Loss of endothelial Foxp1 increased AngII-mediated cardiac fibrosis, which was accompanied by myofibroblast proliferation and endothelial-mesenchymal transition (endMT) with elevated expression of fibrotic matrix genes. TGF-β signal pathway was identified as Foxp1 direct downstream target gene in endothelial cells to regulate the fibrosis. Increased AngII-mediated cardiac hypertrophy was observed when loss of endothelial Foxp1, with elevated cardiac hypertrophic genes and fetal myosin gene (myh7) expression but reduced adult mature myosin gene (myh6) expression via endothelial ET-1 signal. The pathological remodel in endothelial Foxp1 deficient mutant mice finally led to worsen cardiac diastolic dysfunction as shown by elevated E/e’ ratio in echocardiogram. Moreover, the similar pathological remodel and worsen cardiac diastolic and systolic dysfunction as shown by reduction of LV fractional shortening (FS) and ejection fraction (EF) were observed in pressure overload transverse aortic constriction (TAC) model when loss of endothelial Foxp1.

Conclusions:
This study uncovered a previously unrecognized anti-fibrotic and anti-hypertrophic role of Foxp1 in pathological cardiac remodel, which is achieved through the regulation of endothelial TGF-β/ET-1 signals, implying the promising therapeutic targets in pathological cardiac remodeling diseases and heart failure.
Abstract 523: The Effects of Spontaneous Exercise on Murine Model of Deep Vein Thrombosis
Authors: Palmer Olivia R , Diaz Jose A , Greve Joan M
Published: March 2019

Introduction:
Recent studies suggest physical activity may have beneficial effects on deep vein thrombosis (VT). Long-term moderate exercise has been shown to reduce inflammatory cytokines and attenuate plasma fibrinogen compared to uninterrupted sitting. However, the effect exercise may have in VT is unknown. Our objective is to quantify the effects of prophylactic exercise on the thrombotic process in a murine model of VT.

Methods:
C57BL6 male mice were either provided in-cage running wheels (E: n=6) starting 2 weeks prior to and continuously following VT induction, or no wheel exposure (C: n=4). VT was induced with the electrolytic inferior vena cava model (EIM), which induces thrombosis in the continuous presence of blood flow. MR images were acquired days 2 (acute) or 6 (chronic) post VT using a T2-weighted sequence sensitive to edema-related fluid, then the thrombi were harvested for histology.

Results:
Four of six mice engaged in exercise. Three of four mice continued running after thrombus induction, with reduced activity (88 ± 11% reduction). While thrombus volume was not significantly different between groups, the volume of thrombus exhibiting high T2 signal was reduced by ~57% at day 6 in exercising mice, suggesting a decreased inflammatory response with exercise (C 0.81 ± 0.13 mm
3
vs. E 0.35 ± 0.07 mm
3
,
p
=0.08). Fibrin content, assessed by Picro-Mallory staining, was reduced by 60% at day 2 in exercising mice (C 2.08 ± 0.29 um
2
vs. E 0.83 ± 0.45 um
2
,
p
=0.09).

Conclusion:
Our results suggest that exercise may reduce inflammation in VT and lead to less fibrin-rich clots. Future studies will increase the number of animals, investigate inflammatory components, and extend to more chronic time points. Understanding the impact of exercise on specific thrombotic components could lead to better recommendations for patients with DVT.
Abstract 533: Gold Nanoparticles Allow CT Imaging of Experimental Atherosclerosis
Authors: Kosuge Hisanori , Nakamura Maki , Oyane Ayako , Tajiri Kazuko , Murakoshi Nobuyuki , Sakai Satoshi , Sato Akira , Aonuma Kazutaka
Published: March 2019

Background:
Macrophages play a key role in the development of atherosclerosis, and are important targets for the detection of vascular inflammation. Gold nanoparticles (GNPs) are widely used as contrast agents for computed tomography (CT) due to high X-ray attenuation. We evaluated GNPs for imaging of macrophages, both
in vitro
and in murine carotid arteries.

Methods:
Mouse macrophage cells (RAW 264.7) were incubated with GNPs (AuroVist
TM
15 nm, Nanoprobes, Yaphank, NY) at doses of 0, 50, 100 μg Au/ml for 24 hours. Cells were scanned with micro-CT imaging system (LaTheta LCT-100A, Hitachi-Aloka, Tokyo) for cellular uptake. Cell viability was assessed by MTT assay. FVB mice (N=3) induced diabetes and hyperlipidemia were developed macrophage-rich atherosclerotic lesions by left carotid ligation (operation group). After 2 weeks, mice were injected with GNPs (2 mice: 10 mg Au/mouse, one mouse: 20 mg Au/mouse) via tail vein and scanned with micro-CT imaging system at 24 hours after injection. One mouse without induction of hyperlipidemia and diabetes and carotid ligation (control group) was also scanned after injection of GNPs (10 mg Au/mouse).

Results:
The CT values of macrophage cells incubated with GNPs were significantly higher compared to those of Au-free cells (p<0.02) (Figure 1). Macrophages incubated with GNPs showed a similar viability as control cells (Figure 2). The CT values of a ligated left carotid artery injected with 20 mg Au was higher compared to those of a ligated left carotid with 10 mg Au, a non-ligated left carotid of a control, and non-ligated right carotids (operation group and control) at 24 hours after injection (Figure 3).

Conclusion:
CT imaging can detect macrophages with GNPs
in vitro
and accumulation of higher dose of GNPs in vascular lesion. GNPs have the potential as a contrast agent for imaging of atherosclerosis.
Abstract 534: Aging is Associated with Peroxide Mediated Increased Thrombin Generation
Authors: Kumar Rahul , Sonkar Vijay , Wegman-Points Lauren , Sharathkumar Anjali , Pierce Gary L , Dayal Sanjana
Published: March 2019

Human aging is associated with increased incidence of deep vein thrombosis (DVT), but the mechanisms are not fully elucidated. Using experimental models of DVT, we and others have demonstrated that mice also display increased susceptibility to thrombosis during aging. Further, we established that aged mice overexpressing antioxidant glutathione peroxidase (Gpx1; that converts peroxides to water) are protected from age associated increased susceptibility to DVT. However, the mechanistic pathway for peroxide-dependent thrombosis remains elusive. Recent studies have suggested that hydrogen peroxide mediated activation of neutrophils causes release of neutrophil extracellular traps (NETs). NETs are known to activate coagulation and contribute to the development of DVT. We hypothesized that aging cause peroxide-dependent increased release of NETs and increased thrombin generation. We studied Gpx1 Tg mice and their wild type (WT) littermates at 4 (young) and 20 months (old) of age. Cell-free DNA (cfDNA), a circulating marker of NETs was measured in the plasma using Qubit assay. We observed a significant increase in cfDNA in old WT mice compared to young WT mice (28.2±2.2 vs. 11.4±0.8 ng/μl, P<0.001), but there was no difference in cfDNA between old WT and old Gpx1 Tg mice. Next, we assessed thrombin generation using a Calibrated Automated Thrombogram (Daignostic Stago). The aged WT mice showed significant increase in endogenous thrombin potential (ETP, 430.2±26.7 vs. 277.9±42.1 nM.min, P<0.05) as well as peak amplitude, compared to young WT mice (65.4±4.3 vs. 40.8±7.1 nM, P<0.05). Interestingly, ETP was significantly decreased in aged Gpx1 Tg mice (P<0.05 vs. aged WT), suggesting a peroxide mediated effect on thrombin generation with aging. To examine how the findings in aged mice translate to human aging, we examined plasma samples from healthy young (18-39 years, n=11) and older (50-79 years, n=17) human subjects. We observed a significant increase in cfDNA and ETP in older compared to younger humans (13.4±.9 vs. 12.3±.4 ng/μl, p<0.05 for cfDNA and 1917.6±141.2 vs 1475.9±66.3 nM.min, p<0.05 for ETP). These data suggest that both mouse and human aging is associated with increased release of cfDNA and higher ETP, and that in mice peroxide mediates effects of aging on thrombin generation.
Abstract 535: Proteomics Identifies Endothelium-derived Clusterin as a Novel Regulator of Von Willebrand Factor Multimerization
Authors: Luo Jincai
Published: March 2019

Background:
Secretion of vascular endothelial cells (ECs) is considered to be the first line of defense against vascular injury. Vascular endothelial growth factor (VEGF) is one of the most important endothelial growth factors, involved in various endothelial functions including the regulatory secretion of ECs. However, the systematic analysis of the VEGF-induced secretory proteins has not yet been carried out.

Objectives:
To investigate the secretory proteins rapidly released from ECs under VEGF stimulation and their functions on the ECs.

Methods:
A non-serum culture system was established for analyzing the proteins secreted form human primary ECs in response to VEGF with mass spectrometry.

Results:
In the proteomic result, 362 proteins were identified and assigned to the VEGF-induced secretome, including the known von Willebrand factor (VWF), a multimeric protein mediating adhesion of platelets at vascular injury sites. Clusterin, a multifunctional protein involved in various diseases including Alzheimer disease and cancers, was selected for further study as a new VEGF-induced secretory protein. Clusterin was co-localized with VWF both in Weibel-Palade bodies (WPBs) and extracellular VWF strings of ECs. We showed that the localization of clusterin in pseudo-WPBs in 293T cells depends on the carboxy-terminal domains of VWF. Functional study showed that deficiency of clusterin in ECs increased the size of VWF multimers.

Conclusions:
We not only obtained a VEGF-induced secretome using primary ECs, but also determined clusterin as a novel component of WPBs, which is capable of maintaining the normal pattern of VWF multimers, and suggest its function on vascular thrombosis and haemostasis.
Abstract 549: Improvement in Large Density HDL Particle Number by NMR is Associated with Reduction in Coronary Soft Plaque Burden by Coronary Computed Tomography Angiography in Psoriasis
Authors: Elnabawi Yousssef A , Dey Amit K , Goyal Aditya , Groenendyk Jacob W , Genovese Leonard D , Belur Agastya D , Rodante Justin A , Chen Marcus Y , Bluemke David A , Remaley Alan T , Playford Martin P , Mehta Nehal N
Published: March 2019

Background:
Patients with psoriasis (PSO), a chronic inflammatory disease associated with dysfunctional lipoprotein profile and accelerated risk of MI, have increased burden of subclinical atherogenesis by coronary computed tomography angiography (CCTA). Large HDL particle (l-HDLp) number by NMR has been shown to associate negatively with cardiovascular (CV) events independent of traditional lipoprotein levels. We hypothesize that increase in l-HDLp would inversely associate with soft plaque volume (PV) as well as non-calcified coronary burden (NCB).

Methods:
Consecutive treatment naïve PSO patients (n= 92 arteries) underwent CCTA (320 detector row, Toshiba) at baseline and one-year. Soft plaque volume and non-calcified burden were assessed using a semi-automated software (QAngio, Medis). Lipoprotein profiling and cholesterol efflux capacity was done by NMR.

Results:
Patients were middle aged and at low CV risk by traditional risk scores (Table 1). With improvement of PSO severity at one year and no change in traditional CV risks, l-HDLp increased (5.8 ± 0.4 vs. 6.3 ± 0.5, p=0.007) concurrently with cholesterol efflux capacity (0.95 ± 0.02 vs 1.01 ± 0.02, p=0.003). PV reduced at one-year (3.7 ± 1.2 vs. 2.9 ± 1.1 mm
3
, p=0.01), as well as NCB (1.20 ± 0.06 vs. 1.07 ± 0.06 mm
2
, p<0.001). Coronary burden was inversely associated with l-HDLp beyond traditional CV risk factors (β = -0.47, p<0.001).

Conclusions:
Increase in l-HDLp was inversely associated with coronary plaque burden possibly through improvement in HDL function via cholesterol efflux capacity, suggesting a role for NMR lipoprotein profiling in assessment of CVD in chronic inflammatory states.
Abstract 551: Is Free Cholesterol Bioavailability a Determinant of Dysfunctional, Atherogenic High Density Lipoproteins? Refining the Model of Reverse Cholesterol Transport
Authors: Gillard Baiba K , Yelamanchili Dedipya , Gotto Antonio M , Rosales Corina , Pownall Henry J
Published: March 2019

Although cardiovascular disease (CVD) negatively correlates with high-density lipoprotein-cholesterol (HDL-C) levels, to date HDL-raising therapies have not reduced CVD; thus, in the context of reverse cholesterol transport (RCT), HDL quality, i.e., functionality, may be more important to atheroprotection than HDL quantity. The current RCT model comprises free cholesterol (FC) efflux from macrophages to apo AI to give nHDL, LCAT-mediated nHDL-FC esterification forming mature HDL, SR-B1-mediated hepatic uptake of HDL lipids, followed by sterol metabolism and excretion. Our recent studies challenge this model: We observed that nHDL apo AI, FC and phospholipid (PL) metabolically segregate. In mice, plasma nHDL FC and PL are hepatically cleared with t
1/2
~3 min; nHDL-apo AI is cleared more slowly with t
1/2
= 460 min. FC esterification is 100X slower, and thus a minor RCT step. These results and the observation that nHDL is FC-rich (~64 mol%) led to a revised model of RCT, with a focus on FC bioavailabilty rather than CE uptake. HDL from SR-B1
-/-
mice is also FC-rich and associated with atherosclerosis. Moreover, the magnitude of HDL-C content/particle is associated with carotid artery atheroprogression (Qi et al JACC 2015 65:355-363), and HDL from hyperlipidemic HIV patients, which are at increased CVD risk, also have a high mol% FC compared to controls (29 vs 16 mol%, p<0.05). Thus, we hypothesize that high HDL-FC bioavailability, measured as the product of mol% HDL-FC and HDL particle number, is a metric for dysfunctional, atherogenic HDL. We studied SRB1
-/-
mice, which are atherosusceptible and a model of dysfunctional HDL. Compared to WT mice, SR-B1
-/-
mice have a higher HDL particle number and mol% FC (~58 vs. 15). Compared to WT HDL, SRB1
-/-
HDL is more resistant to disruption by GdmHCl and serum opacity factor, indicating a resistance to remodeling. We plan to compare FC bioavailability of WT and SRB1
-/-
HDL according to the kinetics of HDL-FC transfer to LDL. Our studies will determine if increased HDL-FC bioavailability in dysfunctional FC-rich HDL supports whole-body hypercholesterolemia that could increase CVD risk.