bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 489: Disruption of HuR-mediated VEGF-A mRNA Stabilization as a Mechanism of Age-related Defects in Arteriogenesis
Authors: Mantsounga Chris S , Healy Abigail , Ceneri Nicole , Berus Joshua , Watts Jerome , Chun Hyung , Choudhary Gaurav , Morrison Alan R
Published: March 2019

Aging and age-related diseases like peripheral arterial disease are associated with impaired inflammatory arteriogenesis in response to injury. Macrophage expression of pro-inflammatory and pro-angiogenenic factors can be altered during aging. We seek to define the molecular mechanism that determine the impact of aging on macrophage-dependent arteriogenesis. Recently, we defined a critical signaling pathway that involves CCL2 stimulation coupled to ICAM-1 adhesion, resulting in rapid nuclear-to-cytosolic translocation of the RNA-binding protein, HuR, with its consequent stabilization of VEGF-A mRNA. Preliminary data from bone marrow-derived macrophages (BMDMs) demonstrated decreased VEGF-A level by both mRNA and protein in aged mice (52-week old) when compared to young (10-week old). BMDMs from aged mice also revealed increased markers of senescence. The hypothesis is that age-related changes in arteriogenesis are a conseqeuence of altered HuR-mediated VEGF-A stabilization. In a hindlimb ischemia model of arteriogenesis, we found reduced flow recovery in aged mice. Moreover, small arterial angiography by microCT confirmed decreased arteriogenesis. Loss of functional arteriogenesis was associated with decreased muscle tissue VEGF-A levels despite adequate macrophage recruitment to the muscle tissue. BMDM HuR expression was unaffected in aged mice, and CCL2-coupled ICAM-1 adhesion demonstrated comparable nuclear-to-cytosolic translocation in aged BMDMs, indicating the signaling of HuR activation remained intact. However, BMDMs demonstrated a loss of HuR binding to VEGF-A mRNA with consequent decreased VEGF-A mRNA half-life. In summary, HuR-mediated VEGF-A mRNA stabilization and consequent inflammatory-mediated arteriogenesis is disrupted in aged animals.
Abstract 507: Dedicator of Cytokinesis 2 Regulates Smooth Muscle Cell Proliferation via p38 Mitogen-Activated Protein Kinase Signaling Pathway
Authors: Guo Xia , Li FeiFei , Wang Yung-Chun , Dong Kun , Chen Shi-You
Published: March 2019

Smooth muscle cell (SMC) proliferation is an important process during vascular development and involved in vascular remodeling of a number of cardiovascular diseases including atherosclerosis, hypertension, and restenosis after angioplasty. However, the underlying mechanisms are not completely understood. In the present study, we identified dedicator of cytokinesis 2 (DOCK2) as one of the factors mediating the SMC proliferation. Knockout of DOCK2 (DOCK2-/-) caused abnormal blood vessel development in yolk sac and hemorrhage in mouse embryos. In addition, dorsal aorta wall structure was disrupted in DOCK2-/- embryos, and the SMC numbers in the dorsal aorta were decreased by 49% compared with wild type (WT). Consistently, proliferating cell nuclear antigen (PCNA)-positive cells in DOCK2-/- dorsal aorta media were much less the WT. These data suggest that the abnormal vascular development in DOCK2-/- embryos was due to the defective SMC proliferation. In vitro, platelet-derived growth factor (PDGF)-BB and serum upregulated DOCK2 along with PCNA expression in SMCs. Knockout or knockdown of DOCK2 by its shRNA inhibited the PDGF-BB-induced PCNA expression while forced expression of DOCK2 induced PCNA expression in SMCs. 5-ethynyl-2'-deoxyuridine (EdU) assay showed that knockdown of DOCK2 blocked PDGF-BB-induced SMC proliferation. Mechanistically, DOCK2 induced SMC proliferation by activating p38 mitogen-activated protein kinase (MAPK). Overexpression of DOCK2 induced while knockdown of DOCK2 inhibited p38 phosphorylation. Blockade of p38 MAPK signaling by its pathway-specific inhibitor diminished DOCK2-induced SMC proliferation. In a rat carotid artery balloon-injury model, knockdown of DOCK2 by adenoviral delivery of its shRNA blocked the injury-induced neointima formation and attenuated the PCNA expression. Furthermore, deletion of DOCK2 blocked ligation-induced neointima formation and PCNA expression in mouse arteries. Taken together, our studies demonstrated that DOCK2 is an important factor regulating SMC proliferation during embryo development as well as vascular remodeling in pathological conditions.
Abstract 512: Bcl11b is a Newly Identified Regulator of Vascular Smooth Muscle Function and Stiffness
Authors: Valisno Jeff Arni , Elavalakanar Pavania , Nicholson Christopher , Singh Kuldeep , Avram Dorina , Cohen Richard A , Mitchell Gary F , Morgan Kathleen G , Seta Francesca
Published: March 2019

Background:
B-cell leukemia 11b (Bcl11b) is a zinc-finger transcription factor known as master regulator of T lymphocytes and neuronal development during embryogenesis. However, a role for Bcl11b in the cardiovascular system has never been described. Based on human findings from a genome-wide association study (GWAS) that a gene desert region downstream of
BCL11B
, known to function as
BCL11B
enhancer, harbors single nucleotide polymorphisms (SNPs) associated with increased arterial stiffness, we sought to examine relations between Bcl11b and arterial function.

Methods and Results:
We found for the first time that Bcl11b is expressed in the vascular smooth muscle (VSM) of human and murine vasculature and transcriptionally regulates the expression of VSM contractile proteins smooth muscle myosin and smooth muscle α-actin. Lack of Bcl11b in VSM-specific Bcl11b null mice (BSMKO) resulted in an increased expression of Ca
++
-calmodulin-dependent serine/threonine phosphatase calcineurin in BSMKO VSM cells and aortas, which were inversely correlated with levels of phosphorylated VASP
S239
, a calcineurin de-phosphorylation target. Decreased pVASP
S239
in BSMKO aortas was associated with increased actin polymerization (F/G actin ratio), consistent with pVASP
S239
’s function as regulator of cytoskeletal actin rearrangements, and was normalized by treatment with calcineurin inhibitor cyclosporine A. Functionally, Bcl11b deletion in VSM cells translated in increased aortic force, stress and wall tension, measured ex vivo in BSMKO aortas in organ baths, and increased pulse wave velocity, the
in vivo
index of arterial stiffness, in BSMKO mice compared to WT littermates. Moreover, Bcl11b and pVASP
S239
expression were decreased in aortas of obese and aged mice, two models of arterial stiffness. Bcl11b deletion in VSM had no effect on baseline blood pressure or angiotensin II-induced hypertension, measured in conscious WT and BSMKO mice by radiotelemetry, but dramatically increased the incidence of angII-induced aortic aneurysms in BSMKO mice.

Conclusions:
Taken together, our results identify VSM Bcl11b as a novel and crucial regulator of VSM cell phenotype and vascular structural and functional integrity
Abstract 520: Skeletal Muscle Myosin Supports the Anticoagulant Functions of Activated Protein C and Protein S but not Protein C Activation by Thrombin:Thrombomodulin
Authors: Heeb Mary J , McDowell Olivia , Yamashita Atsuki , Fernandez Jose' A , Mosnier Laurent O , Deguchi Hiroshi , Griffin John H
Published: March 2019

Skeletal muscle myosin was reported by Deguchi et al to promote thrombus formation ex vivo for flowing human whole blood, to support prothrombinase activity in plasma and to promote prothrombin activation by factors (F) Xa and Va in purified systems. Skeletal muscle myosin binds FXa and FVa and effectively replaces the procoagulant activity of phospholipid vesicles. We therefore assessed whether myosin might replace phospholipids in support of the anticoagulant function of activated protein C (APC) and its cofactor protein S (ProS) for inactivation of purified FVa. When purified component prothrombinase assays were used to monitor FVa inactivation by APC, FVa activity was significantly reduced by APC in the presence of myosin compared to controls, and FVa inactivation in the presence of 40 nM myosin over 25 min was much greater than that in the presence of 40 nM phospholipids and similar to the rate of FVa inactivation in the presence of 25 μM phospholipids (see Figure). Inhibition of the APC-enhancing effects of myosin were confirmed to be specific for myosin because the anti-myosin inhibitor, myoVin-1, inhibited myosin’s effects. ProS at ≥ 25 nM enhanced the myosin-stimulated inactivation of FVa by APC. The ProS anticoagulant enhancement effect was most obvious at lower APC concentrations, as expected. Immunoblots showed that myosin enhanced Arg506 cleavage in FVa by APC alone; but notably in the presence of ProS, myosin enhanced both Arg506 and Arg306 cleavages in FVa. Myosin did not enhance activation of protein C by thrombin in the presence or absence of thrombomodulin. Thus, in purified reaction mixtures, skeletal muscle myosin remarkably can support both procoagulant thrombin generation, as previously reported, and compensatory anticoagulant mechanisms in the presence of APC and ProS, as shown here. Further studies are needed to determine the molecular basis for these reactions and to assess the physiologic and pathologic relevance of these reaction mechanisms.
Abstract 530: Hemostasis Revisited: a Combined Light and Electron Microscopy Analysis of Hemostatic Plug Formation
in vivo
Authors: Tomaiuolo Maurizio , Matzko Chelsea N , Poventud-Fuentes Izmarie , Weisel John W , Brass Lawrence F , Stalker Timothy J
Published: March 2019

We have previously shown in the microcirculation that gradients of platelet agonists develop during the hemostatic response as a consequence of the solute transport properties within the evolving platelet mass. These agonist gradients result in the formation of a gradient of platelet activation extending from the site of vascular injury. Here we utilized a combined light and electron microscopy approach to examine the organization of hemostatic plugs formed in response to larger injuries that result in significant bleeding in the mouse jugular vein. The results show that the hemostatic response to vessel wall puncture in a large vein is similarly characterized by a gradient of platelet activation. Specifically, we found that 1) platelets are the primary cellular component of hemostatic plugs in large veins; 2) platelet activation is heterogeneous, with fully activated and degranulated platelets present at the interface of the damaged vessel wall and extending to the extravascular side of hemostatic plugs, while platelets aggregated on the luminal side of plugs were minimally activated; 3) fibrin, and by extension thrombin activity, is localized primarily in the extravascular space; 4) P2Y
12
antagonism impairs platelet aggregation, resulting in a prolonged bleeding time when the hole created by the puncture injury does not completely fill with platelets; and 5) the importance of P2Y
12
signaling in establishing a hemostatically competent plug is related to the size of the hole through which blood escapes the vasculature. Taken together, these results provide novel insights into the mechanisms of hemostatic plug formation in vivo, demonstrate in detail the mechanism by which P2Y
12
antagonists result in bleeding, and highlight differences in the composition and morphology of hemostatic plugs versus pathologic thrombi.
Abstract 553: Scavenging Reactive Aldehydes With 5'-o-pentyl-pyridoxamine (PPM) Improves HDL Function and Reduces Atherosclerosis in Ldlr Deficient Mice
Authors: Huang Jiansheng , Zhang Linda , Yancey Patricia , Tao Huan , Ding Lei , Zhang Youmin , Oates John , Amarnath Venkataraman , Roberts Jackson , Davies Sean , Linton MacRae F
Published: March 2019

Background:
Lipid peroxidation products impair the cholesterol efflux capacity of high-density lipoprotein (HDL) and contribute to the development of atherosclerosis. The effect of inhibition of HDL dysfunction by scavengers on HDL function and whether scavenging reactive aldehydes with PPM protects against the development of atherosclerosis was examined.

Methods and Results:
HDL of familial hypercholesterolemia (FH) subjects have impaired ability to promote cholesterol efflux and FH-HDL contain 5-fold more malondialdehyde adducts (MDA) than control HDL. In vitro studies revealed that reactive aldehyde MDA crosslinks apolipoprotein AI (apoAI) and impairs the ability of HDL to promote cholesterol efflux from
Apoe
-/-
macrophages. 5'-O-pentyl-pyridoxamine (PPM), a potent scavenger of reactive aldehydes, abolished MDA-mediated crosslinking of apoA-I in HDL by 80 % (P<0.05). PPM prevents the reduction in cholesterol efflux capacity of MDA treated HDL in Apoe-/- macrophages. Furthermore, PPM significantly improved the cholesterol efflux capacity of HDL from

Ldlr
-/-

mice fed a Western diet (WD) for 16 weeks (P<0.05), indicating that PPM protects HDL from modifications by reactive aldehydes and maintains HDL function in vivo. Importantly, administration of 1 mg/mL of the reactive aldehyde scavenger PPM, versus 1 mg/mL of the nonreactive analogue PPO, to

Ldlr
-/-

mice consuming a WD for 16 weeks reduced the extent of proximal aortic atherosclerosis by 45% (P<0.05). Immunohistochemistry studies revealed that PPM reduced the macrophage content and the number of TUNEL positive cells by 55% (P<0.05) and by 60% (P<0.01) in advanced atherosclerotic lesions of

Ldlr
-/-

mice, respectively. In addition, the necrotic core area was reduced by 52% (P<0.05) in advanced atherosclerotic lesions in

Ldlr
-/-

mice treated with PPM compared to the control group.

Conclusions:
Treatment with PPM, a reactive aldehyde scavenger: 1) inhibits MDA-ApoA1 adduct formation thereby preserving HDL cholesterol efflux capacity in

Ldlr
-/-

mice; 2) reduces the number of apoptotic cells in atherosclerotic lesions and the necrotic core area in lesions of

Ldlr
-/-

mice. These results support the therapeutic potential of PPM in the treatment of atherosclerotic cardiovascular disease.
Abstract 562: Foamy Monocytes in Hypertriglyceridemia
Authors: Peng Xueying , Lian Zeqin , Perrard Xiaoyuan D , Dong Henry , Mukherjee Aparna , Zhu Haibo , Ballantyne Christie M , Wu Huaizhu
Published: March 2019

Objective:
Hypertriglyceridemia (HTG) increases risk for atherosclerotic cardiovascular disease, but the mechanisms remain poorly defined. Foamy monocytes are lipid-loaded monocytes in circulation that contribute to atherosclerosis under hypercholesterolemia. Human study has proved that HTG is associated with formation of foamy monocytes. Our study is to examine formation of foamy monocytes in HTG and their potential contribution to atherosclerosis in mouse models.

Approach and results:
In vivo mouse models of HTG included wild-type C57BL/6 mice on high fat diet (HFD) injected intraperitoneally with LPL inhibitor, Poloxamer 407 (P407, 0.25mg/g, every two days), as a chemically-induced model and mice with transgenic overexpression of human ApoCIII (ApoCIIItg) as a genetic model. Based on CD11c and CD36, monocytes were identified as CD36
-
CD11c
-
, CD36
+
CD11c
-
and CD36
+
CD11c
+
subsets. In the first model, at 24h of the first injection, triglyceride levels increased to 367 ±84 mg/dL, higher than that of control group with saline injection (60 ±22 mg/dL, n=4, P<0.001). Meanwhile, the side scatter (SSC, representing cell granularity) values and Nile red staining for lipids of CD36
+
CD11c
+
monocytes increased significantly, indicating formation of foamy monocytes, in mice with HTG. Furthermore, CD11c mean fluorescence intensity of CD36
+
CD11c
+
foamy monocytes increased significantly at 2 weeks of P407 injection. In ApoCIIItg mice fed HFD (5 weeks), the percentage and SSC value of CD36
+
CD11c
+
monocytes increased significantly (37%±5%, 247±8), also indicating elevated granularity and lipid accumulation of these monocytes, compared to wild-type mice (26%±3%, p<0.05; 226±8, p<0.05, n=4-6). In vitro treatment with human triglyceride-rich lipoprotein (hTGRL) for 24h increased the granularity and Nile red staining intensity of THP-1 monocytes, indicating foamy monocyte formation. hTGRL treatment also increased THP-1 monocyte expression of CD36, with greater uptake of cholesteryl ester-rich lipoprotein.

Conclusion:
High triglyceride promotes foamy monocyte formation and induces monocyte phenotypic changes in mice and tissue culture, with increased expression of CD11c and CD36, which may contribute to development of atherosclerosis under HTG.
Abstract 582: Vascular Smooth Muscle-derived Macrophage are a Major Source of MCP1 in Atherosclerosis
Authors: Owsiany Katherine , Nguyen Anh , Owens Gary K
Published: March 2019

Smooth muscle cells (SMC) are conventionally thought to promote atherosclerotic plaque stability by investing within the fibrous cap and protecting the blood flow from the harmful inflammatory plaque core. However, lineage tracing studies have shown that SMC in plaque can undergo a Klf4-dependent transition
in vivo
to a macrophage-like state, characterized by loss of SMC markers and expression of multiple macrophage markers, lipid accumulation, and phagocytic activity. Of major interest, SMC induced to the macrophage state in vitro by cholesterol loading also show increased production of the atheropromoting cytokine MCP1 (Monocyte chemoattractant protein 1). These results are surprising since it has been largely assumed that monocyte derived macrophages are the major source of MCP1 and that this acts to exacerbate lesion pathogenesis by inducing recruitment of additional circulating monocytes. Using MCP1-mCherry reporter mice on an ApoE
-/-
background, we show that MCP1 is highly expressed by medial and fibrous cap SMC. Further, we show that SMC-specific KLF4 knockout mice, which show marked reductions in the frequency of SMC-derived macrophages, show a 50% decrease in MCP1 immunostaining in late stage atherosclerotic lesions, suggesting that these cells are a major source of MCP-1 and produce it via a Klf4-dependent mechanism. Consistent with this possibility, we demonstrated that SMC-specific tamoxifen conditional MCP1 knockout mice on an ApoE
-/-
background (Myh11-CreERT2 eYFP ApoE
-/-
MCP1-mCherry), showed greatly decreased total MCP1 protein level within the aorta. We are currently completing examination of the effects of SMC specific MCP1 KO on late stage lesion pathogenesis in our Myh11-CreERT2 eYFP ApoE
-/-
MCP1-mCherry mice following 18 weeks of Western diet feeding. Taken together results provide evidence that SMC derived cells are an unexpected major source of MCP1 within lesions.
Abstract 586: Oxidized LDL Induce a Metabolic Switch Through CD36 in Macrophages
Authors: Chen Yiliang , Huang Wenxin , Yang Moua , Silverstein Roy
Published: March 2019

Under atherogenic conditions, low-density lipoproteins are converted to oxidized forms (oxLDL) by reactive oxygen species. They are then recognized by scavenger receptors such as CD36 in macrophages and stimulate pro-inflammatory functions of those innate immune cells. Although oxLDL are believed to contribute to chronic inflammation during atherosclerosis development, how the inflammatory status is sustained is not well understood. In this study, we explored how oxLDL affects the metabolic status of macrophages, which is increasingly being recognized as a key regulator of macrophage phenotypes.

Exposure of wild type macrophages to oxLDL, but not native LDL, suppressed mitochondria oxidative phosphorylation (OXPHOS) as measured by oxygen consumption rate (OCAR), and increased glycolysis as measured by extracellular acidification rate (ECAR), in a dose-dependent manner. At 50μg/ml oxLDL for 24h there was a 79% reduction in OXPHOS and 60% elevation in glycolysis. Exposure to 50μg/ml oxLDL increased glucose uptake by 1.4 fold. Inhibition of glycolysis with 2-deoxy-glucose blocked oxLDL-induced HIF-1α expression as well as cytokine production, linking glycolytic metabolism to the pro-inflammatory response to oxLDL. These metabolic changes were not seen in
cd36
null macrophages.

In conclusion, oxLDL induce a metabolic switch in macrophages from mitochondrial OXPHOS to glycolysis through CD36-mediated signaling. This metabolic switch is important for inflammatory activation of macrophages and may be an underlying mechanism for sustained inflammatory status of those cells in the context of atherosclerosis.
Abstract 588: Corticotropin-releasing Hormone via Its Type2 Receptor Inhibits Insulin-stimulated NO Formation in HUVECs
Authors: Chung Ick-Mo , Jin Hai Dan , Kim Hyang
Published: March 2019

Background:
Stress may provoke endothelial dysfunction thereby inducing atherosclerotic cardiovascular diseases (ASCVD). Corticotropin-releasing hormone (CRH) is recognized as a key regulator of hypothalamo-pituitary-adrenal axis under stress, however, its role in endothelial function and ASCVD is not known well. Insulin can produce NO formation in endothelial cells through eNOS activation. We studied effect of CRH and urocortin 2 (UCN2) on insulin-stimulated NO formation in HUVECs.

Methods and Results:
HUVECs were incubated in serum-free media for 8 h prior to the experiment. Treatment of HUVECs with insulin (100 nM) enhanced NO production detected by 5 μM 4,5-diaminofluorescein-diacetate. Fluorescent and phase contrast images were obtained using an fluorescence microscope (Em max : 515 nm), using an exposure time based on control conditions, and mean fluorescence intensity was calculated with ImageJ. Insulin-stimulated NO production was markedly inhibited by either CRH or UCN2 (100 nM each), which was reversed mostly by 100 nM astressin 2B (CRH receptor type 2 (CRHR2) inhibitor) and partly by 100 nM NBI35965 (CRHR1 inhibitor). CRH induced Rho-associated kinase (ROCK) and p-IRS-1(Ser). Insulin (2nM) stimulated p-eNOS (Ser1177), which was inhibited by CRH.

Conclusions:
Taken together, CRH/UCN2 via mainly its type 2 receptor signaling can inhibit insulin-stimulated eNOS/NO pathway. This CRH’s inhibitory effect on insulin signaling is mediated at least partly by activation of ROCK/ p-IRS-1 (Ser).
Abstract 604: Anti-apoptotic Role of Leptin in Adipose Tissue
Authors: Zhang Yuebo , Somers Virend K , Dong Yu , Singh Prachi
Published: March 2019

Background:
Leptin contributes to obesity-related cardiometabolic pathology. While the role of adipose tissue dysfunction in altering metabolic profile in obesity is recognized, the direct effects of leptin signaling in adipose tissue are not completely understood. Among factors impacting adipose tissue function in obesity, adipocyte death is associated with increased inflammation with consequent adipose tissue insulin-resistance. Furthermore, apoptosis of adipocyte progenitor cells (preadipocytes) is important as it lowers adipose tissue capacity to maintain and expand fat storage.

Objective:
To determine the effects of leptin on regulation of proteins important to apoptotic pathways in cultured human white preadipocytes (HWP) and differentiated HWP (dHWP).

Methods:
Cells were exposed to increasing concentrations of leptin to evaluate the role of leptin on regulation of anti-apoptotic Bcl2, and pro-apoptotic Bax and CD95 expression. Next, we examined if pre-exposure to leptin attenuated TNF-α and FasL induced apoptosis.

Results:
Leptin upregulated transcription of Bcl2 (p=0.02) and down-regulated transcription of Bax (p=0.02) and CD95 (p=0.04) in a leptin-concentration dependent manner in dHWP. Leptin dependent increases in Bcl2 (p=0.004) and decreases in Bax (p=0.003) and CD95 (P=0.03) protein expression were also observed. Furthermore, 24 hour pre-exposure to leptin protected cells from TNF-α (p=0.01; p=0.006) and FasL (p=0.03; p=0.007) induced apoptosis in dHWP and HWP respectively. Additionally, leptin attenuated TNF-mediated activation of the p53 pathway, suggesting another mechanism by which leptin mediates its inhibitory effects on TNF-induced apoptosis.

Conclusion:
Leptin regulates the expression of proteins involved in the apoptotic pathway such that it decreases the apoptotic potential of adipose tissue by increasing anti-apoptotic Bcl2 and decreasing pro-apoptotic Bax and CD95 protein expression. In contrast to our in-vitro findings, prior studies in adipose tissue from obese humans report decreased Bcl2 and increased Bax protein expression, along with high leptin expression. This suggests that increased adipose tissue apoptosis in obesity may at least in part be secondary to altered leptin signaling.
Abstract 606: The Differential Roles of the Adaptor Proteins Nck1 and Nck2 in Shear Stress-Induced Endothelial Activation
in vitro
and
in vivo
Authors: Alfaidi Mabruka , Green Jonette M. , Orr A. Wayne
Published: March 2019

Hemodynamic shear stress critically regulates endothelial activation and atherogenesis by affecting cytoskeletal dynamics and endothelial gene expression. The Nck adaptor proteins (Nck1 and Nck2) couple tyrosine kinase signaling to induction of cytoskeletal remodeling pathways, and we previously demonstrated that a peptide corresponding to a Nck-binding sequence in p21 activate kinase (PAK) blunts shear stress-induced proinflammatory signaling (PAK, NF-κB) and permeability
in vitro
and
in vivo
. However, the specific roles of Nck1 and Nck2 in flow-induced endothelial activation remain to be elucidated. Here, we demonstrate that Nck1, but not Nck2, critically regulates shear stress-induced endothelial activation. We show that Nck1 deficiency (siRNA knockdown, genetic knockout) decreases basal and shear stress-induced proinflammatory signaling (PAK2, NF-κB) and proinflammatory gene expression (ICAM-1, VCAM-1). In addition, only Nck1, but not Nck2, knockdown/knockout limited shear-induced endothelial permeability. However, other shear stress-dependent pathways, such as Akt activation, proved Nck1-independent. By contrast, selective Nck2 depletion paradoxically increased basal PAK2 activation without significant effects on NF-κB signaling or permeability. Using the partial carotid ligation model of disturbed flow, we found that Nck1 knockout mice showed significantly reduced proinflammatory gene expression that was not further diminished upon Nck2 deletion. However, Nck2 was not inconsequential to the flow response, as Nck2 knockdown or knockout reduced actin cytoskeletal alignment with laminar flow. Taken together, our data suggests that Nck1 plays a dominant role in flow-induced endothelial activation in response to shear stress, whereas Nck2 may critically regulate flow-induced cytoskeletal remodeling and endothelial alignment.
Abstract 614: From Genotype to Phenotype: Molecular and Functional Characterization of the CVD-associated SNP rs11574 in
ID3
Authors: Henderson Christopher A , Lipinski Michael , Zhou Andrea , Garmey James , McNamara Coleen A
Published: March 2019

Atherosclerosis is the primary pathological process of CVD and is strongly controlled by heritable factors; however, the mechanisms and pathways through which many of these genetic components regulate plaque development are poorly understood. Recently, a SNP in the coding region of the gene inhibitor of differentiation 3 (
ID3
) at rs11574 was identified in multiple studies to be associated with CVD. Mutation of rs11574 from the major allele (G) to the minor allele (A) changes the 105
th
amino acid of ID3 from an alanine to a threonine and is associated with an attenuated ability of ID3 to antagonize bHLH transcription factors and to prevent them from binding to DNA and activating transcription.

The current study utilized co-immunoprecipitation to demonstrate that the minor allele of rs11574 specifically impairs the ability of ID3 to bind to and sequester E12 and no other member of the bHLH family. ChIP studies further demonstrated that the impaired regulatory ability of the ID3 minor allele variant increases E12 occupancy at promoter regions and enhances transcription of E12 target genes including smooth muscle alpha actin (
Acta2
) and p21 in murine vascular smooth muscle cells (VSMCs). To study the role of this SNP in human cells in intact chromatin under endogenous regulation, we genome edited human 293T cells using CRISPR/Cas9 to produce the allelic variants of rs11574 in the
ID3
gene (GG, AG, and AA). Edited cells showed significant changes in cellular proliferation, gene expression, and promoter occupancy depending upon genotype at rs11574. Cells containing the minor allele of rs11574 displayed significantly increased p21 expression and proliferated more slowly than cells containing the major allele of rs11574; whereas, cells heterozygote at rs11574 displayed an intermediate phenotype. These data implicate rs11574 in the regulation of cellular proliferation and mature VSMC marker expression, key phenotypes regulated in the processes of lesion development and formation.
Abstract 628: Overexpression of ABCA1 in Cultured Endothelial Cells Using Helper-Dependent Adenovirus Enhances ApoAI-Mediated Cholesterol Efflux and has Anti-Inflammatory Effects
Authors: Stamatikos Alexis , Dronadula Nagadhara , Ng Philip , Palmer Donna , Knight Ethan , Wacker Bradley , Tang Chongren , Kim Francis , Dichek David
Published: March 2019

Background:
ABCA1 removes cholesterol from vascular wall cells via apoAI-mediated efflux, generating HDL that transports cholesterol to the liver for excretion. This process of reverse cholesterol transport is atheroprotective; therefore, strategies that increase vascular wall ABCA1 may prevent or reverse atherosclerosis. Cholesterol efflux mediated by ABCA1 and apoAI can also have anti-inflammatory effects; however, excess depletion of cellular cholesterol can cause cell stress and apoptosis. We tested whether transducing endothelial cells (EC) with a helper-dependent adenoviral vector that expresses ABCA1 (HDAdABCA1) enhances apoAI-mediated cholesterol efflux and reduces inflammatory markers without causing cellular toxicity.

Methods:
We cloned rabbit ABCA1, constructed HDAdABCA1, transduced bovine aortic EC (BAEC) with either HDAdABCA1 or empty vector (HDAdNull). We measured ABCA1 protein by immunoblotting and apoAI-mediated cholesterol efflux by loading EC with
3
[H] cholesterol, then adding apoAI protein to serum-free medium. We assessed EC phenotype using MTT (metabolic activity), BrdU (proliferation), wound-healing assay (migration), and flow cytometry (apoptosis). We measured ICAM-1, VCAM-1, IL-6, and TNFα mRNA in transduced EC by qRT-PCR both under basal conditions and after serum-starvation, addition of apoAI, and LPS challenge.

Results:
We observed a ~3-fold increase in ABCA1 protein in EC transduced with HDAdABCA1 and a ~2-fold increase in apoAI-mediated cholesterol efflux (P<0.01 for both). This level of ABCA1 overexpression did not alter EC metabolic activity, proliferation, migration, or apoptosis. Under basal conditions, HDAdABCA1 had no effect on inflammatory markers. However, after serum starvation and addition of apoAI, HDAdABCA1-transduced EC had reduced expression of inflammatory markers both before and after LPS treatment.

Conclusions:
HDAdABCA1 increases EC ABCA1 expression and enhances apoAI-mediated cholesterol efflux, but does not cause toxicity or increase inflammatory markers. In contrast, ABCA1 overexpression appears to have anti-inflammatory effects. Future studies will test if HDAdABCA1 decreases lipid rafts, and whether overexpression of ABCA1 in EC in vivo is atheroprotective.
Abstract 632: Myeloid beta catenin Deficiency Exacerbates Atherosclerosis in Low-density Lipoprotein-receptor Deficient Mice
Authors: Wang Fang , Liu Zun , Park Se-Hyung , Gwag Taesik , Lu Weiwei , Ma Murong , Sui Yipeng , Zhou Changcheng
Published: March 2019

Objective:
The Wnt/β-catenin signaling is an ancient and evolutionarily conserved pathway that regulates essential aspects of cell differentiation, proliferation, migration and polarity, and canonical Wnt/β-catenin signaling has also been implicated in the pathogenesis of atherosclerosis. Macrophage is one of the major cell types involved in the initiation and progression of atherosclerosis, but the role of macrophage β-catenin in atherosclerosis remains elusive. This study aims to investigate the impact of β-catenin expression on macrophage functions and atherosclerosis development.

Approaches and Results:
To investigate the role of macrophage canonical Wnt/β-catenin signaling in atherogenesis, we generated low-density lipoprotein receptor-deficient (LDLR
-/-
) mice with myeloid-specific β-catenin deficiency. As expected, deletion of β-catenin decreased macrophage adhesion and migration properties in vitro. However, deficiency of β-catenin significantly increased atherosclerosis in LDLR
-/-
mice without altering the plasma lipid levels. Mechanistic studies revealed that β-catenin can regulate activation of signal transducer and activator of transcription (STAT) pathway in macrophages, and ablation of β-catenin resulted in STAT3 downregulation and STAT1 activation, leading to elevated macrophage inflammatory responses and increased atherosclerosis.

Conclusions:
This study demonstrates a critical role of myeloid β-catenin expression in atherosclerosis by modulating macrophage inflammatory responses.
Abstract 644: Some Perspectives of Preventive Cardiology Development
Authors: Ghevondyan Tigran H
Published: March 2019

Background:
The role of preventive cardiology becomes more significant in solving heart problems. The analysis of our research results allows making assumptions, promising for prevention of coronary atherosclerosis (CA).

Material and Methods:
The studies were carried out on: hearts of humans died from accidents, from acute myocardial infarction; myocardial biopsies, heart surgical material of patients; hearts of animals; insects flying muscles; placentas of human and animals. The methods were used: autopsy, planimetry of atherosclerotic lesions of aorta and main coronary arteries, heart biopsy, microangio-x-ray, histology, stereology, experimental modeling, measuring of hemodynamic parameters, micromechanography, electron microscopy, cytochemistry, mathematical modeling, statistics.

Aim:
To show that existing data may serve as a basis for further development of new ways to prevent CA.

Results:
Huge differences of myocardial arterial bed (MAB) density exist. The richness of MAB correlates directly with life expectancy of people. In hearts with scant MAB CA develops earlier and atherosclerotic lesions are more severe. Therefore scant MAB is considered as an intracardial risk factor (IRF) for IHD. Large individual differences exist in human hearts also for values for the duration of myocardial fibers oxygen supply. The long time is another IRF for IHD. The density of intraorganic arterial bed in human placentas varies widely. There is a positive correlation between placental arterialization density and newborn weigh. The following morphological multifactorial systemic measurement methods are developed: a) for heart muscle oxygen supply; b) for contractility of cardiomyocyte using small heart muscle biopsies (patented); c) for gas exchangeability of placenta. The efficiency and exactness of each method were examined.

Conclusion:
The received data allow assuming that atherosclerotic profile of human heart CA will be possible to foretell on the next day of born. In cases of bad prognosis a plan of enrichment of MAB, a plan for spatial improvement of the “capillary - myocardial fiber” system may be composed and undertaken. The received data allow in advanced future to draw up a project of new heart with necessary preset structural parameters.
Abstract 649: Skeletal Muscle Satellite Cells Play a Role in Regulating Angiogenesis and Arteriogenesis
Authors: Hansen Laura M , Liu Wenxue , Joseph Giji , Taylor W. Robert
Published: March 2019

Satellite cells are myogenic stem cells that play a critical role in skeletal muscle repair by proliferating and differentiating into myoblasts to repair muscle fibers. However, their role in reestablishing vascular supply following injury is not well defined. We hypothesized that satellite cells promote vascular growth through paracrine signaling induced following muscle injury or ischemic damage from diseases such as peripheral artery disease. We used a murine model of hind limb ischemia and found that satellite cells increased 3.4 fold (p<0.01) in response to this ischemic insult. We used a co-culture system to determine that satellite cells led to a 3.5 fold and 2.8 fold increase in smooth muscle and endothelial cell migration (p<0.0001). These results demonstrate the satellite cells produce paracrine factors which drive cell migration required for both angiogenesis and arteriogenesis. To test the potential therapeutic capability, alginate encapsulated satellite cells were delivered in the hind limb ischemic model. We found the encapsulated cells were viable for up to 2 weeks and mice that received satellite cells had significantly increased perfusion (28%, p<0.05) at 2 weeks as measured by Laser Doppler imaging and a 1.9 fold (p<0.5) increase in capillaries and small vessels measured by histological staining. To examine the role of satellite cells in a physiological setting and determine if they are critical to robust recovery in our model, we used a Cre-Lox system in which recombination results in the production of diphtheria toxin a (DTA) to deplete Pax7 specific cells. Mice which lacked satellite cells had decreased perfusion by Laser Doppler imaging (p<0.05) and capillary density compared animals with intact satellite cells. A cytokine array and genomics analysis show that several factors related to angiogenesis and cell migration are upregulated in satellite cells in response to ischemia. In conclusion, we have found that satellite cells proliferate in response to ischemia, produce factors that drive cell migration, and their delivery or depletion affect vascular growth in vitro. We believe that our studies show that satellite cells play an important role vascular growth and are a novel potential therapy for peripheral artery disease.
Abstract 660: Elevated Calcium Drives Inflammatory-Driven Angiogenesis
Authors: Mulangala Jocelyne , Akers Emma J , Solly Emma L , Psaltis Peter J , Tan Joanne T , Nicholls Stephen J , Bursill Christina , Di Bartolo Belinda A
Published: March 2019

Background:
Peripheral arterial disease (PAD) is characterised by accelerated arterial calcification and impairment in angiogenesis, both of which can influence cardiovascular clinical outcomes. Studies implicate calcification as a driver of PAD, however, the mechanisms by which calcification modulates angiogenesis remain poorly understood. This study assessed the effect of high calcium on angiogenesis both
in vitro
and
in vivo
.

Methods:
Human Coronary Artery Endothelial Cells (EC) were cultured and treated with calcification medium (CM) (CaCl
2
2.7mM, Na
2
PO
4
2.0mM) for 24 h. Angiogenic assays of proliferation, migration and tubulogenesis were conducted, and immunoblotting assessed angiogenic regulatory proteins.
In vivo
studies employed a calcification model with 8-12-week-old male OPG
-/-
and wildtype (C57BL6/J, control) mice which underwent hind-limb ischaemia (HLI) surgery. Blood flow reperfusion was assessed by Laser Doppler Perfusion Imaging (LDPI). Calcium assay assessed calcium levels in blood serum of the C57BL6/J and OPG
-/-
mice.

Results:
CM significantly reduced EC tubulogenesis and viability (34% and 58% p<0.05) but increased migration (p<0.0001) over 24h. There was a significant increase in the protein levels of angiogenic regulators VEGFA (p<0.0001) and HIF-1α (p<0.0001). CM also significantly increased NF-κB (P65) nuclear protein, a key mediator of inflammatory-driven angiogenesis. qRT-PCR showed upregulation of osteoinductive factor Bone Morphogenetic protein (BMP2), and transcription factor Runx2 mRNA expression, both involved in osteogenesis and calcification. Blood serum calcium levels were significantly increased in OPG deficient mice. LDPI found there was significantly reduced blood-flow reperfusion in OPG
-/-
mice at days 6 (0.03±0.01 vs 0.32±0.05 p<0.01) and day 10 (0.08±0.01 vs 0.35±0.05 p<0.01) post HLI induction, compared to controls.

Conclusion:
This is the first demonstration that high-levels of calcium impair ischaemia-driven angiogenesis
in vivo
and cause inflammation in ECs that suppresses tubule formation
in vitro
, despite upregulation of key angiogenic regulators VEGFA and HIF-1α. These findings have implications for the development of therapies that can suppress calcification in PAD.
Abstract 661: Regulation of
CCL2
Expression in Vascular Endothelial Cells by a Long Noncoding RNA
Authors: Khyzha Nadiya , Khor Melvin , Hedin Ulf , Maegdefessel Lars , Wilson Michael D , Fish Jason E
Published: March 2019

Vascular inflammation is a critical driver of chronic diseases such as atherosclerosis. A network of NF-κB-dependent leukocyte adhesion molecules and chemokines are induced in endothelial cells (ECs) in response to inflammatory mediators. This includes chemokine (C-C motif) ligand 2 (
CCL2
), which contributes to atherosclerosis by recruiting monocytes to the endothelium. Recently, long noncoding RNAs (lncRNAs) have been implicated in regulating gene expression through epigenetic mechanisms, but lncRNAs remain poorly studied in the context of vascular inflammation and NF-kB pathway regulation. LncRNAs are frequently retained in the nucleus where they interact with chromatin remodelling complexes to modulate the expression of neighboring protein-coding genes. Hence, identifying NF-kB-regulated neighboring mRNA-lncRNA pairs in vascular endothelial cells may uncover functional lncRNAs that play a role in fine-tuning the expression of their neighboring inflammatory genes.

The Arraystar human lncRNA microarray V3 was employed to identify differentially expressed lncRNAs and mRNAs in ECs stimulated with the pro-inflammatory cytokine, IL-1β. Neighboring IL-1β-regulated mRNA-lncRNA pairs demonstrated a larger magnitude of mRNA induction than mRNAs lacking a neighboring lncRNA. This phenomenon was associated with shared regulatory elements and localization within the same topologically associated domain. Follow-up analysis was performed on the nuclear-enriched,
lncRNA-CCL2
, which is transcribed through a super-enhancer near
CCL2
. Both
lncRNA-CCL2
and
CCL2
responded to the same inflammatory stimuli. Similar to
CCL2
,
lncRNA-CCL2
transcript was elevated in unstable human atherosclerotic plaques. Knockdown of
lncRNA-CCL2
decreased
CCL2
mRNA levels in multiple EC cell lines, but had no effect on other inflammatory genes or distal
CCL
genes. Hence, our approach has uncovered neighboring IL-1β-regulated mRNA-lncRNA pairs and identified a novel functional lncRNA,
lncRNA-CCL2
.
Abstract 664: Regulation of Angiogenesis and Associated Inflammation by Antioxidant Glutathione in Smooth Muscle Cells
Authors: Shrestha Bandana
Published: March 2019

Peripheral artery blockage due to formation of atherosclerotic plaques in PVD (Peripheral Vascular Diseases) causes loss of blood flow to peripheral limbs leading to pathogenic consequences, necrosis and in severe cases amputation of the limb. Angiogenesis, the development of new collateral blood vessels is necessary to compensate for this loss of blood flow and prevent pathogenesis resulting from blockage. However, PVD patients, to their detriment, often see decreased angiogenesis and lack of adequate compensatory neo-vascularization along with decreased circulating antioxidant levels. Angiogenesis requires a specific balance of Reactive Oxygen Species(ROS) and neutralizing antioxidants including Glutathione (GSH). Interestingly, our mouse models of PVD with femoral artery ligation (FAL) show a contradictory increase in angiogenesis with a slight decrease in GSH (~80% GSH, Gclm HET mice) compared to WT (100% GSH), not seen with severely decreased GSH in Gclm KO mice (~20% GSH). This suggests that the balance of ROS and antioxidants can be adjusted to a favorable outcome. This led us to hypothesize that a slight decrease in GSH is beneficial and provides “necessary” inflammation to kick-start angiogenesis. To investigate this hypothesis, we performed mRNA-seq analysis on Smooth Muscle Cells (SMCs), an important component of blood vessels known to promote angiogenesis. SMCs were isolated from Gclm WT/HET/KO mice and subjected to hypoxia/re-oxygenation (HR) to mimic blood flow blockage and reperfusion
in-vivo
. Our hypothesis was confirmed with an increased inflammatory gene expression profile in HET SMCs compared to WT and KO. Results show that slight decrease in antioxidant GSH leads to significant increases in the IL-8, NF-kB, fMLP, MAPK kinase and Notch related signaling pathways, all of which positively influence inflammation associated angiogenesis. Our study suggests that a slight increase in oxidative stress leads to an “appropriate” increase in inflammation and provides adequate environment for accelerated angiogenesis and subsequent tissue perfusion.
Abstract 666: Klotho Suppresses the Protein Kinase R-mediated Inflammatory Response to Soluble Matrilin-2 in Human Aortic Valve Interstitial Cells
Authors: The Erlinda , Ao Lihua , Zhai Yufeng , Zhang Peijian , Fullerton David A. , Meng Xianzhong
Published: March 2019

Calcific aortic valve disease (CAVD) is a chronic, progressive inflammatory disease. Soluble extracellular matrix (ECM) proteins can function as damage-associated molecular patterns (DAMPs) and may play a role in the progression of CAVD. Matrilin-2 is an ECM protein and has been found to up-regulate the pro-osteogenic activity in human aortic valve interstitial cells (AVICs). Klotho is an anti-aging protein that is recently found to have an anti-inflammatory effect. The impact of matrilin-2 and Klotho on AVICs inflammatory response is unclear. This study is to test the hypothesis that matrilin-2 induces the inflammatory response in human AVICs and to explore the anti-inflammatory potential of Klotho for suppression AVIC inflammation.

Methods and Results:
Human AVICs isolated from normal valves were treated with recombinant matrilin-2 (2.0 μg/ml). Matrilin-2 caused NF-κB-dependent increase in the levels of ICAM-1, MCP-1 and IL-6. In addition, matrilin-2 induced rapid activation of PKR through Toll-like receptor (TLR) 2 and 4. Treatment with PKR inhibitors, 2-AP or C13H8N4OS, prior to matrilin-2 stimulation, abrogated NF-κB phosphorylation and intranuclear translocation. Inhibition of PKR abolished the production of inflammatory mediators induced by matrilin-2 in human AVICs. Further experiments using recombinant Klotho revealed that Klotho (0.5 μg/ml) suppressed the activation of PKR and NF-κB, and markedly reduced the production of inflammatory mediators in human AVICs exposed to matrilin-2.

Conclusion:
This study demonstrates that soluble matrilin-2 induces the inflammatory response in human AVICs through a TLR-PKR-NF-κB signaling cascade and that Klotho is capable of suppressing human AVICs inflammatory response to a soluble ECM protein. The novel findings of this study indicate that soluble ECM proteins may fuel the progression of CAVD by inducing aortic valve inflammation and that Klotho has the potential for suppression of such inflammation.