bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 211: MKL1 Affects Proliferation/Survival Property of Pro-Atherogenic Macrophages
Authors: An Jianbo , Naruse Taeko K , Kimura Akinori
Published: March 2019

Coronary artery disease (CAD) is one of the leading causes of death in the most countries. Atherosclerosis of coronary arteries often results in high incidence of vascular occlusion and is recognized as the major cause of CAD. We have previously reported from a genome-wide association study that a promoter SNP in megakaryoblastic leukemia 1 gene (
MKL1
), which conferred higher transcriptional activity, was significantly associated with the susceptibility to CAD as well as coronary atherosclerosis. MKL1 is a Rho-Rock signaling-responsive co-activator of serum response factor, and regulates a variety of cellular functions. We found that MKL1 was highly expressed in activated macrophages in the neointima of atherosclerotic lesions from human and ApoE-knockout (KO) mouse. In addition, the expression level of MKL1 in lesional macrophages was increased during the development of atherosclerosis in ApoE-KO mice.

We have established a transgenic mouse line, MKL1-TgM, in which human
MKL1
was specifically overexpressed in monocyte/macrophage lineage cells. In this study, MKL1-TgM was crossbred onto ApoE-KO background. ApoE-KO/MKL1-TgM fed with normal chow developed severer atherosclerosis and showed poorer survival rate than ApoE-KO mice. Serum level of pro-inflammatory cytokines was increased in ApoE-KO/MKL1-TgM. Interestingly, ApoE-KO/MKL1-TgM also developed hypertriglyceridemia, which was associated with severe lipodystrophy.

We also investigated how MKL1 regulated macrophages function. Recently, local proliferation/accumulation of lesional macrophages was reported to be a crucial event in atherosclerosis. We treated bone marrow-derived macrophages (BMDMs) with stimuli, such as M-CSF or OxLDL, and found that proliferation property of BMDMs from MKL1-TgM was elevated. On the other hand, BMDMs from MKL1-TgM were more resistant to apoptosis. Furthermore, immunohistochemistry analyses of lesional macrophages in ApoE-KO/MKL1-TgM showed enhanced proliferation and mitigated apoptosis.

Taken together, our data indicated that MKL1 would contribute to the development of atherosclerosis by modulating pro-atherogenic functions of macrophages, providing a potential molecular target for the therapy and prevention of atherosclerosis.
Abstract 212: Chronic Systemic Inflammation Accelerates Endothelial Cholesterol Crystal Formation and Lysosomal Dysfunction
Authors: Baumer Yvonne , Sanda Gregory , Teague Heather , Dagur Pradeep , Ng Qimin , Harrington Charlotte , Varghese Nevin , Sorokin Alexander , Bleck Christopher , Rodante Justin , Kruth Howard , Joshi Aditya , Dey Amit , Playford Martin , Mehta Nehal
Published: March 2019

Objective:
Atherogenesis is an inflammatory process accelerated in psoriasis, a chronic inflammatory skin disease associated with premature cardiovascular diseases (CVD). Cholesterol crystal (CC) formation is implicated at onset and progression of atherosclerotic CVD, and we have recently shown CC formation to be upregulated in a murine model of psoriasis-related atherosclerosis. Therefore, we investigated inflammatory CC production in this psoriasis mouse model and also using psoriasis patient derived samples.

Methods:
K14-Rac1V12
psoriasis mouse aortas were examined for CC formation and structural abnormalities, such as subendothelial spaces, via electron microscopy and histology. Human aortic endothelial cells (HAoEC) were stimulated with sera from healthy and psoriatic volunteers (n=5 each), inflammatory markers TNFα+IFNγ, LDL or combination of TNFα+IFNγ+LDL. The effect on CC formation and lysosomal function was determined by advanced microscopy and flow cytometry. HAoECs were also treated with TNFα+IFNγ+LDL combined with β-cyclodextrin (CD), a stimulant for cholesterol release from cells, and analyzed for CC formation and lysosomal function.

Results:
Psoriatic mouse aortas showed enlarged subendothelial spaces and 40% increased CC formation under normolipidemic conditions compared to littermate controls. Treatment of HAoECs with psoriasis patient-sera (severe skin disease, low CVD risk score) or TNFα+IFNγ+LDL showed a 2-fold increase in CC formation and 30% increased lysosomal frequency compared to controls. Despite an increase in lysosome frequency, TNFα+IFNγ+LDL treatment decreased overall pH-related lysosomal activity. CD treatment diminished TNFα+IFNγ+LDL-induced CC formation (compared to vehicle), and restored lysosomal function without affecting lysosome frequency.

Conclusion:
Psoriasis mice displayed accelerated CC formation in the absence of hyperlipidemic conditions suggesting chronic systemic inflammation accelerates CC formation
in vivo
and
in vitro
, accompanied by lysosomal dysfunction in ECs. These findings suggest that dyslipidemia and systemic inflammation in psoriasis are important contributors to early atherogenesis and should be treatment targets for CVD risk mitigation.
Abstract 223: Monocytes/macrophages Are Sensitized to Secondary Oxidative Insult by Cardiovascular Toxic Drugs via the P90rsk-erk5 S496 Phosphorylation Pathway, Aggrandizing Atherosclerotic Plaque Formation
Authors: Kotla Sivareddy , Le Nhat-Tu , Singh Meera V. , Ko Kyung Ae , Heo Kyung-Sun , Fujii Yuka , Vu Hang T , McBeath Elena , Thomas Tamlyn , Wang Yin , Gi Young Jin , Tao Yunting , Medina Jan , Stirpe Nicole E. , Lu Wang , Tyrell Alicia , Gates Kathleen J. , Qui Xing , Fujiwara Keigi , Maggirwar Sanjay B. , Schifitto Giovanni , Abe Jun-ichi
Published: March 2019

Increased cardiovascular (CV) events in HIV patients and cancer survivors and the fact that combination antiretroviral therapy (cART) and chemotherapy induce similar unwanted CV effects are becoming evident, but the reason for this is unclear. In this study, we examined the role of p90RSK in the CV toxicity of cART and anti-cancer drugs. The level of p90RSK activation by H2O2 was higher in peripheral monocytes from cART-treated patients than in those from untreated patients. Multiple linear regression analysis involving HIV+/-, Reynolds CV risk score, and basal p90RSK activation revealed HIV+/- and basal p90RSK activation to be strong determinants for plaque formation when adjusted for other independent variables. In murine macrophages most of the cART and several chemotherapy agents activated p90RSK and reduced antioxidant molecule expression and telomere (TL) lengths. p90RSK-mediated ERK5 S496 phosphorylation inhibited transcriptional activity of ERK5 and NRF2, decreasing efferocytosis, antioxidant production and TL lengths. NRF2 activator normalized cART-induced sensitization of p90RSK to H2O2. Lastly, we generated myeloid cells-specific wild type (WT) and dominant negative (DN) p90RSK transgenic mice, and ERK5 S496A knock-in mice, crossed with
Ldlr-/-
mice or treated with a single injection of adeno-associated virus vector (AAV) encoding a gain-of-function mutant of PCSK9 and fed a high fat diet. Our animal studies showed the crucial role of p90RSK-mediated ERK5 S496 phosphorylation in suppressing effecrocytosis and antioxidant production, and up-regulating senescence and inflammation-related molecules expression, leading to atherogenesis. Our results taken together show that p90RSK is activated by anti-HIV and anti-cancer drugs, and this activation sensitizes the monocyte/macrophages to the secondary oxidative insult by reducing NRF2 transcriptional activity and TL length. The p90RSK activation also reduces macrophage efferocytosis and antioxidant capacity, and increases inflammation and senescence via up-regulating ERK5 S496 phosphorylation, thereby accelerating atherosclerosis. Monocyte/macrophage p90RSK-ERK5 S496 phosphorylation could be a good target to prevent drugs-induced CV diseases.
Abstract 228: Multi-omics Mapping Generates a Molecular Atlas of the Aortic Valve and Reveals Networks Driving Disease
Authors: Schlotter Florian , Halu Arda , Goto Shinji , Blaser Mark C , Body Simon C , Lee Lang H , Higashi Hideyuki , DeLaughter Daniel M , Hutcheson Joshua D , Vyas Payal , Pham Tan H , Rogers Maximillian A , Sharma Amitabh , Seidman Christine E , Loscalzo Joseph , Seidman Jonathan G , Aikawa Masanori , Singh Sasha A , Aikawa Elena
Published: March 2019

Background:
No pharmacological therapy exists for calcific aortic valve disease (CAVD), which confers a dismal prognosis without valve replacement. The search for therapeutics and early diagnostics is challenging since CAVD presents in multiple pathological stages.

Methods:
A total of 25 human stenotic aortic valves obtained from valve replacement surgery were analyzed by multiple modalities, including transcriptomics and global unlabeled and tandem-mass-tagged proteomics by liquid chromatography-mass spectrometry.

Results:
Global transcriptional and protein expression signatures differed between the non-diseased, fibrotic, and calcific stages of CAVD, with consistent trends in gene and protein expression across disease stages. Anatomical aortic valve microlayers exhibited unique proteome profiles that were maintained throughout disease progression, and revealed GFAP as a specific marker of valvular interstitial cells (VICs) from the spongiosa layer. CAVD disease progression was marked by an emergence of smooth muscle cell activation, inflammation, and calcification-related pathways. Proteins overrepresented in the disease-prone fibrosa are functionally annotated to fibrosis and calcification pathways, and we found that,
in vitro
, fibrosa-derived VICs demonstrated greater calcification potential than those from the ventricularis. These studies confirmed that the microlayer-specific proteome was preserved in cultured VICs, and that VICs exposed to TNAP-dependent and TNAP-independent calcifying stimuli had distinct proteome profiles, both of which overlapped with that of the whole tissue. Network analysis of protein-protein interaction networks found a significant closeness to multiple inflammatory and fibrotic diseases.

Conclusions:
A spatially- and temporally-resolved multi-omics and systems biology strategy identifies the first molecular regulatory networks in CAVD, a cardiac condition without a pharmacological cure, and describes a strategy for endophenotype characterization that is broadly applicable to comprehensive omics studies of cardiovascular diseases.
Abstract 229: Small GTPase Rap1 deficiency Accelerates Development of Atherosclerosis in ApoE Deficient Mice
Authors: Singh Bandana , McIntyre Tara , Lakshmikanthan Sribalaji , Kaul Sushma , Sorci -Thomas Mary , Chrzanowska-Wodnicka Magdalena
Published: March 2019

Rap1, a ubiquitously expressed small GTPase, integrates signals from multiple receptors and promotes activation and signaling by cell adhesion receptors.
In vivo
, endothelial (EC) Rap1 is required for normal vessel formation, angiogenesis and dynamic regulation of EC barrier. Two Rap1 isoforms, Rap1A and Rap1B, share 95% identity, and EC deletion of both isoforms leads to embryonic lethality due to cardiovascular defects. We have recently demonstrated that Rap1 is a novel regulator of shear sensing in endothelium and that it is essential for nitric oxide release, and that Rap1 deficiency leads to EC dysfunction. These findings demonstrated that Rap1 plays an essential role in vasoprotective response of endothelium in response to shear stress. The objective of this study is to determine the specific role of Rap1 response to laminar, vasoprotective flow and pro-inflammatory, disturbed flow by examining the effect of EC deletion of Rap1B, the more prominent Rap1 isoform, on progression of atherosclerosis. To investigate the role of Rap1 in progression of atherosclerosis, we crossed apolipoprotein E-deficient (ApoE
-/-
) mice with tamoxifen-inducible, EC specific Rap1B-knockout (Cadh5-CreERT
+/0
; Rap1B
f/f
) to generate atherogenic EC-Rap1B KO (Athero-Rap1B KO) mice. We hypothesized that Rap1B deficiency will exacerbate progression of atherosclerosis. Athero-Rap1B KO and littermate controls (Cadh5-Cre-negative mice or mice injected with carrier oil only) were fed high fat, western diet (21.2% fat, 0.2% cholesterol) for 16 weeks. No difference in total cholesterol levels were observed between Athero- Rap1B KO and control mice. Atherosclerotic lesion formation was visualized with oil O red and plaque area was quantified in en-face preparations. (n=5-9). We observed increased lesion area in the descending aorta of Athero-Rap1B KO mice (0.7351 ± 0.1065 vs 0.4866 ± 0.09812; p =0.0584, one-tailed t-test, Welch corrected) and a strong trend towards larger lesion area in the total aorta (2.142 ± 0.5058 vs 1.267 ± 0.1718; p=0.0679), and the arch of the aorta (1.489 ± 0.4343 vs 0.7807 ± 0.1152; p=0.0747) compared with control mice. These findings demonstrate that Rap1B deficiency accelerates progression of atherosclerosis and show that Rap1 plays a vasoprotective role in both laminar flow and disturbed flow areas.
Abstract 230: Matrix Regulation of Lymphangiogenesis in Atherosclerosis
Authors: Singla Bhupesh , Lin Huiping , Ghoshal Pushpankur , Martel Catherine , Csanyi Gabor
Published: March 2019

Background:
Atherosclerosis is a consequence of an imbalance of cellular uptake of lipoproteins versus removal of cholesterol from the arterial wall. The lymphatic vasculature represents the primary route of cholesterol removal from atherosclerotic vessels. Previous studies have linked matrix protein thrombospondin-1 (TSP1), a well-known inhibitor of angiogenesis, with human atherosclerotic disease; however the mechanisms by which TSP1 contributes to atherosclerosis and its effect on arterial lymphangiogenesis remain unknown.

Methods:
The effect of TSP1 on human lymphatic endothelial cell (LEC) proliferation, migration and tube formation was investigated
in vitro
. TSP1 protein expression was analyzed in human and murine (ApoE
-/-
,
12 weeks Western diet) atherosclerotic aortic tissue and lesion free segments using Western blotting. The role of TSP1 and its cognate receptor CD47 in the pathogenesis of atherosclerosis was investigated using knockout animals.

Results:
TSP1 expression was significantly increased in both human and ApoE
-/-
atherosclerotic arteries compared to plaque free segments in human and wild type aortic tissue. TSP1 at physiologically relevant concentrations (11-22 nM, 24 hrs) inhibited LEC proliferation, migration, and tube formation. RT-PCR data demonstrated that CD47 is the predominant TSP1 receptor expressed in LEC (> 50-fold over CD36 mRNA levels). Finally, genome-wide deficiency in TSP1 and CD47 receptor attenuated atherosclerotic lesion formation in the aorta and brachiocephalic artery by ~50% compared to wild type controls. Importantly, there was no difference in plasma cholesterol levels, body weight, fat and lean mass, fasting plasma glucose, and blood pressure between the experimental groups.

Conclusions:
The present study suggests that matrix protein TSP1 via CD47 receptor contributes to the pathogenesis of atherosclerosis. Inhibition of arterial lymphangiogenesis by TSP1-CD47 signaling may contribute to arterial lipid accumulation and atherosclerosis.
Abstract 234: Nuclear Export of Telomeric Repeat Binding Factor 2 (terf2)-interacting Protein (terf2ip) Expression, One of the Sherterin Complex Molecule, is Crucial for Disturbed Flow-induced Endothelial Inflammation and Senescence via Upregulating Terf2ip S205 Phosphorylation
Authors: Vu Hang T , Le Nhat-Tu , Heo Kyung-Sun , Fujii Yuka , Kolta Sivareddy , Ko Kyung Ae , Wang Yin , Thomas Tamlyn , Fujiwara Elena , Giancursio Carolyn , Fujiwara Keigi , Abe Jun-ichi
Published: March 2019

Atherosclerosis is a multifactorial disease mainly caused by endothelial cell (EC) dysfunction resulting from EC senescence. Both EC inflammation and senescence are increased by disturbed flow (d-flow) but the exact mechanism is still unknown. The potential role of TERF2IP in regulating inflammation has been reported, but it has been controversial, especially in ECs. We found that d-flow-induced NF-kB activation was significantly inhibited by the depletion of TERF2IP, but TNF-induced NF-kB activation under laminar flow (l-flow) was not inhibited by TERF2IP depletion. TERF2IP S205 phosphorylation was increased by d-flow, but not by s-flow. This TERF2IP S205 phosphorylation was crucial for d-flow-induced TERF2IP nuclear export, which also caused nuclear export of its binding partner TERF2 in a piggy-back manner. Both the depletion of TERF2IP (TERF2IP siRNA) and overexpression of TERF2IP S205A mutant inhibited d-flow-induced TERF2 nuclear export as well as subsequent EC senescence. In addition, we found the key role of p90RSK activation in regulating EC inflammation and senescence via TERF2IP S205 phosphorylation. To determine the role of TERF2IP and TERF2 cellular localization in vivo, we performed in vivo en face immunostaining of mouse aortic arch in EC specific TERF2IP knock out (EC-TERF2IP-KO) and control mice using anti-TERF2IP or TEFR2 and anti-VE-cadherin antibodies. In control mice TERF2IP expression was very low in the l-flow area. In contrast we found a significant increase of TERF2IP expression, especially in the extra-nuclear region of ECs in the d-flow area. More nuclear localization of TERF2 was observed in EC-TERF2IP-KO mice than those in wild type, which supports our finding in vitro. Significant decreases of VCAM-1 expression and apoptosis were found in the d-flow area in EC-TERF2IP-KO mice compared to control mice. Lastly, we found a significant decrease in d-flow-induced atherosclerotic plaque formation after partial carotid ligation in EC-TERF2IP-KO mice crossed with Ldlr-/- mice compared to control Ldlr-/- mice. These data suggest the key role of nuclear export of TERF2IP in up-regulating EC inflammation and senescence via p90RSK-mediated TERF2IP S205 phosphorylation.
Abstract 236: Magnetic Resonance T1 High Signal Intensity in Coronary Atherosclerotic Plaques Reflects Intraplaque Hemorrhage-derived Erythrocyte Content
Authors: Yamashita Atsushi , Kuroiwa Yasuyoshi , Uchida Akiko , Maekawa Kazunari , Gi Toshihiro , Imamura Takuroh , Asada Yujiro
Published: March 2019

Objective:
Acute myocardial infarction is triggered by plaque disruption and thrombus formation, therefore, it is important to detect high thrombotic risk plaque non-invasively. Coronary high signal intensity plaque (HIP) on T1 weighted magnetic resonance imaging (T1WI) was associated with future coronary events. The aim of this study is to identify pathological findings reflecting HIPs using hearts obtained from autopsy cases and blood samples.

Approach and Results:
Formalin-fixed hearts (n=7) were imaged with non-contrast T1WI with a 1.5 T magnetic resonance system. We defined HIPs (n=11) or non-HIPs (n=25) as a coronary plaque to myocardial signal intensity ratio (PMR) of √ 1.4 or √ 1.4, respectively. The corresponding histological sections were histologically and immunohistochemically analyzed. Plaque and necrotic core areas or frequency of intraplaque hemorrhage in HIPs were significantly larger or higher than those in non-HIPs. The high intensity portion in large coronary plaque was immunopositive for CD68, CD163, glycophorin A, and fibrin. Glycophorin A-, matrix metalloprotease (MMP) 9-, tissue factor-immunopositive areas were larger in HIPs than those in non-HIPs. Glycophorin A-, fibrin-, MMP9-, and tissue factor-immunopositive areas were positively correlated with PMR. We calculated T1 relaxation time of venous blood, plasma, and erythrocyte-rich blood in vitro. Blood coagulation shortened T1 relaxation time of the blood and plasma, and T1 relaxation times in coagulated whole blood and erythrocyte-rich blood were significantly shorter than those in plasma.

Conclusions:
Majority of coronary HIPs may reflect intraplaque hemorrhage-derived erythrocyte content, and may be a novel marker for plaque instability and thrombogenic potential.
Abstract 238: Biological Role and Therapeutic Potential of Nitric Oxide Signaling in Aortic Valve Stenosis: Insights From Genetic Modulation of DDAH1
Authors: Zhang Bin , Roos Carolyn , Oehler Elise , Verzosa Grace , Arghami Arman , Hagler Michael , Miller Jordan D
Published: March 2019

The balance between local asymmetric dimethylarginine (ADMA) levels and dimethylarginine dimethylaminohydrolase 1 (DDAH1) activity is a major determinant of local nitric oxide synthase activity. Previous studies suggested that increased plasma ADMA levels are strongly associated with the aortic valve stenosis, causal or mechanistic links have yet to be elucidated. Thus, the aim of this study was to investigate whether reduction or overexpression of DDAH1 protects aortic valve dysfunction in mice. To experimentally alter DDAH1 levels, we used low density lipoprotein receptor deficient, apolipoprotein B100-only (LA) mice that were either DDAH1 wild-type (D
+/+
or D
0/0
), heterozygous (D
+/-
), or transgenic/overexpressing (D
Tg/0
) littermates. All mice were fed a western diet for 6 months. Reducing DDAH1 increased plasma ADMA levels (LA-D
+/+
=0.89 ± 0.02 μmol/L, LA-D
+/-
=1.1 ± 0.02 μmol/L, p< 0.05) and blood pressure (LA-D
+/+
= 125 ± 4 mmHg, LA-D
+/-
= 141 ± 3 mmHg, p< 0.05) following 6 months of western diet feeding. Critically, aortic valve function was significantly worse in DDAH1-deficient mice (LA-D
+/+
2.20±0.09 m/sec versus LA-D
+/-
=2.41±0.11 m/sec, p< 0.05) and was associated with higher levels of valve cusp calcification (Alizarin Red, LA-D
+/+
= 10 ± 3% versus LA-D
+/-
= 20 ± 4%). Reciprocally, overexpression of DDAH1 in hypercholesterolemic littermates reduced ADMA levels (LA-D
0/0
= 1.4 ± 0.2 μmol/L, LA-D
Tg/0
= 0.7 ± 0.1μmol/L, p< 0.05) and systolic blood pressure (LA-D
0/0
= 136 ± 6 mmHg, LA-D
Tg/0
= 124 ± 4 mmHg, p< 0.05). Interestingly, aortic valve function was not improved by overexpression of DDAH1 (LA-D
0/0
= 2.36 ± 0.06 m/sec versus LA-D
Tg/0
mice (2.43 ± 0.21 m/sec), nor was valvular calcification. These results suggest that while endogenous levels of DDAH1 expression protect against increases in circulating ADMA, blood pressure, valvular calcification, and valvular dysfunction. Interestingly, overexpression of DDAH1 does not appear to confer additional protection against progression of valvular stenosis. Collectively, our data suggest effective therapeutic harnessing of the DDAH1/ADMA/NOS axis will require targeting of downstream effector molecules—such as oxidation state of soluble guanylate cyclase—known to be pathologically altered and rate limiting in a variety of cardiovascular diseases.
Abstract 241: Impact of Cigarette Smoke, Next Generation Tobacco and Nicotine Products on the Cytotoxic, Oxidative and Pro-Inflammatory Status of THP-1 Cells
Authors: Brunssen Coy , Giebe Sindy , Hofmann Anja , Brux Melanie , Hewitt Katherine , Lowe Frazer , Morawietz Henning
Published: March 2019

Monocytes exhibiting a pro-inflammatory phenotype play a key role in adhesion and development of atherosclerotic plaques. Next generation tobacco and nicotine products (NGPs) are now widely used globally as an alternative to smoking. Little is known about their pro-inflammatory effects on monocytes. We investigated cell viability, anti-oxidant and pro-inflammatory gene and protein expression in THP-1 monocytes exposed to aqueous extracts of conventional cigarettes (CSE), a tobacco heating product (THP) and an electronic cigarette (EC). Pure nicotine was used as additional control. Treatment with CSE reduced cell viability in a dose-dependent manner, whereas all other test agents showed no difference to control. At the highest non-lethal dose of CSE (20%) the following notable mRNA expression changes were observed for CSE, THP and EC respectively, relative to control; HMOX1 (6-fold, <2-fold, <2-fold), NQO1 (3.5-fold, <2-fold, <2-fold), CCL2 (4-fold, 3.5-fold, 2.5-fold), IL1B (4-fold, 3-fold, <2-fold), IL8 (5-fold, 2-fold, 2-fold), TNF (2-fold, 2-fold, <2-fold), CD31 and ICAM1 were below the 2-fold threshold for all products. With respect to protein expression; IL1B (3-fold, <2-fold, <2-fold) and IL8 (3.5-fold, 2-fold, 2-fold) were elevated over the 2-fold threshold, whereas, CD31, ICAM1, TNF and CCL2 were below 2-fold expression for all products. At higher doses, greater inductions were observed with all extracts; however NGP responses were typically lower than CSE. In conclusion, anti-oxidative and pro-inflammatory processes were activated by all products. NGPs showed similar or lower responses relative to controls than CSE exposed cells.
Abstract 254: The Bromo and Extraterminal Domain Protein (BET) Family Drives Endothelial-To-Mesenchymal Transition and Contributes to Vein Graft Stenosis
Authors: Zhang Mengxue , Wang Bowen , Urabe Go , Guo Lian-Wang
Published: March 2019

Background:
Vein graft bypass remains the most commonly used open procedure for flow-limiting cardiovascular diseases. However, vein grafts are prone to stenosis, which ultimately leads to graft failure (4-year failure rate at ~43%). Previous studies have provided strong evidence supporting that dysfunctional endothelial cells (ECs) of the vein grafts contribute significantly to neoinitma formation (and hence stenosis) by undergoing EndoMT. Our group has recently shown that BRD4, a member of the BET epigenetic reader protein family, is up-regulated in the neointima lesion of stenotic failed vein grafts. Here we have determined whether BRD4, and possibly other BET proteins, would be involved in EndoMT in vein grafts, and ultimately vein graft stenosis/failure.

Method:
For in vitro experiments, we treated rat primary vascular ECs with 100ng/ml TGFβ-1 (to induce EndoMT), and/or BET inhibitors (Pan inhibitor: JQ1; Domain-selective inhibitors: Olinone and RVX208), and/or siRNAs for BRD2, 3, and 4. For in vivo experiments, we used a cuff-based technique to establish jugular vein to carotid artery interposition graft in rats, and lentiviral shRNA delivery to achieve specific knockdown of BRD4 in the graft.

Results:
BET pan-inhibition significantly reduced EndoMT of rat primary vascular ECs in vitro. Similar effects were observed via selective inhibition of bromodomain 2, but not bromodomain 1. Knockdown of BRD4 protein expression in vitro fully recapitulated the inhibitory effect of BET pan-inhibition on EndoMT, whereas knockdown of BRD2 and BRD3 could only lead to partial and marginal abrogation of EndoMT, respectively. In the rat vein graft model, local lentiviral delivery of BRD4 shRNA led to decrease of neointima formation.

Conclusion:
BET proteins, especially BRD4, play an important role in EndoMT of vascular ECs and also neointima formation in vein grafts. Blockage of BRD4 protein could potentially serve as a novel strategy to achieve prolonged patency of vein graft conduits.
Abstract 256: The Direct Characterization of Endothelial Inflammation in Patients with Psoriasis
Authors: Garshick Michael S , Barrett Tessa , Scher Jose , Neimann Andrea , Katz Stuart , Li Xuan , Jelic Sanja , Krueger James , Berger Jeffrey S
Published: March 2019

Objective:
Psoriasis, an inflammatory autoimmune disease, increases the risk of cardiovascular disease (CVD). Active psoriatic disease is linked to systemic vascular inflammation, yet how this contributes to CVD is unknown. Using
in vivo
and
ex-vivo
measures of the vascular endothelium our study investigates the vascular health of psoriasis patients to better understand the mechanism(s) that predispose psoriatics to CVD.

Methods:
Ten patients with active psoriasis (average age 46 years, 50% male (5 of 10), 6% [3.5% – 90%] body surface area involvement) were compared to age- and sex- matched controls.
In vivo
vascular endothelial function was assessed by brachial artery reactivity testing (BART, %) with high resolution ultrasonography. Venous endothelial cells were collected from the brachial vein using guidewires inserted through an angiocatheter and isolated with magnetic beads directed against CD146. Following collection, endothelial RNA was isolated, converted to cDNA and inflammatory gene profiling performed by RT-qPCR with Taqman probes and primers.

Results:
Transcriptomic profiling of venous endothelial cells revealed upregulation of genes associated with inflammatory cytokines and chemokines (
lymphotoxin beta
[2.5 - fold],
CCL3
[3.5 - fold], and
IL-1
β
[2.8 - fold], P < 0.05 for all) and genes related to intracellular adhesion and inflammation (
ICAM1
[2.3 – fold] and
COX-2
[1.4 – fold], P < 0.05 for both) in psoriatics vs. controls. Unexpectedly, endothelial nitric oxide synthase (eNOS) and phosphorylated eNOS (higher levels indicate healthy endothelial NO production) were upregulated (2 - 3 fold) in psoriatics vs. controls (p = 0.24, p = 0.14 respectively). BART was also higher in psoriatics when compared to controls (7.1 ± 1% vs. 3.9 ± 2.7%, P = 0.03).

Conclusion:
This cross-sectional study is the first to directly examine the vascular endothelium of psoriatic patients. Compared to controls, active psoriatic disease was associated with upregulation of cytokines, chemokines and genes regulating intracellular adhesion as well as increased expression of eNOS, and increased BART. These findings suggest potential mechanisms to explain the increased prevalence of atherosclerosis and CVD risk seen in those with psoriasis.
Abstract 257: Apabetalone (RVX-208) Reduces Pathologic Cell-Cell Adhesion and Expression of Key Vascular Inflammation Markers in Monocytes, Endothelial Cells and Mouse Aorta
Authors: Tsujikawa Laura , Wasiak Sylwia , Daze Emily , Sarsons Chris D , Stotz Stephanie C , Jahagirdar Ravi , Studer Deborah , Rinker Kristina D , Sweeney Michael , Johansson Jan O , Wong Norman C , Kulikowski Ewelina
Published: March 2019

Apabetalone (RVX-208) is a bromodomain & extraterminal (BET) protein inhibitor, an epigenetic modifier of gene expression, currently in a phase 3 major adverse cardiac events outcomes trial in post-acute coronary syndrome patients with type 2 diabetes mellitus (DM) (BETonMACE). CVD patients enrolled in phase 2b trials (ASSERT, SUSTAIN and ASSURE) demonstrated a 44% relative risk reduction in cardiovascular disease (CVD) events (Nicholls et al. 2017). In CVD and DM, elevated circulating cytokines potentiate vascular inflammation (VI) through recruitment of leukocytes to the vascular endothelium, which contributes to atherosclerosis and plaque rupture. Previous studies demonstrated that apabetalone has potent anti-inflammatory effects on human aortic endothelial cells (HAEC) and macrophage-like U937 cells. Here we show that TNFα stimulation induced significant adhesion of THP-1 monocytes to inflamed endothelial cells, an outcome reversed by apabetalone treatment under both static (human umbilical vein endothelial cells - HUVEC) and flow (HAEC) conditions. Mechanistically, apabetalone suppressed the TNFα and IL-1β-induced expression of mRNAs of multiple endothelial cell adhesion molecules (CD44, E-selectin, VCAM-1 and MCP-1) and inflammatory cytokines (IL-6, IL-8, IL-1β, and CSF2). Monocytes also respond to TNFα stimulation with an upregulation of inflammatory and adhesion marker expression. In THP-1 cells, apabetalone treatment significantly reduced mRNA expression of CCR1, CCR2, IL-1β, MCP-1, MYD88, TLR4, TNFα, and VLA-4. In the diet-induced obesity (DIO) mouse model that mimics metabolic syndrome, treatment with apabetalone, administered at 150 mg/kg BID for the last 16 weeks of a 22 week study, downregulated aortic adhesion markers (E-selectin and ICAM) and markers of infiltrating immune cells (CCR2 and CD11b). In summary, treatment with apabetalone causes transcriptional changes in monocytes and endothelial cells that translate into a reduction in adhesion under inflammatory conditions. We hypothesize that downregulation of VI by apabetalone may contribute to the reduction in CVD events observed in phase 2 studies. This hypothesis is currently being tested in the ongoing BETonMACE phase 3 trial.
Abstract 258: Palmitate Regulates Diabetic Macrophage Inflammation via the Epigenetic Enzyme JMJD3
Authors: Davis Frank M , Kimball Andrew , Joshi Amrita , Boniakowski Anna , Schaller Matthew , DenDekker Aaron , Kunkell Steven , Moore Bethany , Gallagher Katherine
Published: March 2019

Macrophage (Mϕ) plasticity, allowing for transition of Mϕs from an inflammatory to a reparative phenotype, is critical for normal wound healing. In pathologic conditions, such as type 2 diabetes (T2D), wounds fail to heal due to impaired resolution of inflammation. The mechanism(s) responsible for the persistent inflammatory phenotype in T2D wounds are unclear. Prior studies have shown that the Toll-like receptor (TLR) 4 pathway regulates Mϕ-mediated inflammation in tissues. Growing evidence indicates that TLR4 is a versatile receptor binding a spectrum of ligands including non-microbial ligands such as saturated fatty acids (SFAs). Given the excess SFAs in T2D, the purpose of this study was to examine the role of the SFA palmitate on TLR4 signaling and Mϕ phenotype in diabetic wound healing. We have previously shown that Mϕs isolated from wounds in a murine model of glucose intolerance (diet-induced obesity; DIO) maintained on a 60% high fat diet (HFD) for 12-18 weeks, display increased levels of inflammatory cytokines (IL-1β, IL-12, and TNFα) at both a gene expression and protein level. In the current study, we found that blood monocytes and wound Mϕs from DIO mice display increased TLR4 receptors compared to control blood and wounds. To determine if altered metabolites in the diabetic environment impact Mϕ phenotype, bone marrow derived macrophages (BMDMs) were incubated in serum isolated from DIO or control mice. BMDMs incubated with DIO serum displayed a hyperinflammatory response following LPS stimulation. Further, stimulation with the metabolite palmitate produced significantly increased IL-1β expression in DIO BMDMs compared to controls suggesting that DIO BMDMs are programmed toward an inflammatory response. To determine the mechanism, we examined several epigenetic enzymes known to affect Mϕ polarization and found that palmitate stimulated expression of JMJD3, a histone demethylase, which increases inflammatory gene expression. In conclusion, these studies suggest that the diabetic milieu, specifically increased levels of the SFA palmitate, induces expression of the epigenetic enzyme, JMJD3, in Mϕs and this regulates inflammatory gene expression and cell function.
Abstract 259: S100A9 is a Master Regulator of Inflammation and Repair After Myocardial Infarction
Authors: Marinkovic Goran , DeCamp Lisa , Koenis Duco , Winkler Laura , de Waard Vivian , Nilsson Jan , Jovinge Stefan , Schiopu Aleaxandru
Published: March 2019

Background:
The innate immune response plays an important role in cardiac repair following an acute myocardial infarction. The pro-inflammatory alarmin S100A8/A9 is released in high amounts locally and systemically during acute coronary events. Here, we studied the effects of S100A9 blockade on the innate immune responses involved in post-ischemic myocardial inflammation and repair.

Methods:
We induced MI by permanent left coronary artery ligation in C57BL/6 mice, which were subsequently treated with the specific S100A8/A9 blocker ABR-238901 (30mg/kg) or with buffer for 21 days. Left ventricular function was assessed by echocardiography. Immune cell populations in the myocardium, blood, bone marrow and spleen were analysed by flow cytometry.

Results:
The treatment induced progressive deterioration of the left ventricular systolic function and increased left ventricular volumes in ABR-238901-treated mice compared with controls, suggesting defective repair and negative myocardial remodelling. After 21 days of treatment, infarction size was significantly higher in mice receiving S100A8/A9 blockade (16.1±5.4% vs. 9.9±3.4%, P=0.03). S100A9 blockade inhibited proliferation of hematopoietic stem cells in the bone marrow, and reduced neutrophil and monocyte trafficking from bone marrow and spleen to the circulation and myocardium. Neutrophil and monocyte counts increased in the bone marrow and spleen of mice receiving S100A8/A9 blockade, possibly due to impaired egression. The presence of reparatory CD11b
+
F4/80
+
Ly6C
low
macrophages expressing the efferocytosis receptor MerTK was potently decreased in the myocardium by day 7 post-MI compared to controls (36 820 ± 2 538 vs. 72 371 ± 4 482 cells/heart, P=0.0001). The transcription factor Nur77 mediates phenotype switching from inflammatory Ly6C
hi
monocytes to reparatory Ly6C
lo
macrophages in MI. ABR-238901 lowered the expression of Nur77 in Ly6C
hi/int
monocytes
in-vivo
, total bone marrow derived monocytes
in-vitro
, and the activity of Nur77 in macrophages
in-vitro
.

Conclusions:
S100A8/A9 plays an important role in leukocyte trafficking after MI. Long-term S100A8/A9 blockade impairs efferocytosis and myocardial repair, leading to systolic dysfunction and detrimental remodelling.
Abstract 270: Microsomal Prostaglandin E Synthase-1 and Endothelial EP4 Receptor Reduces Myocardial Ischemia-reperfusion Injury via Improving Microcirculatory Perfusion
Authors: Zhu Liyuan , Xu Chuansheng , Hao Huifeng , Hu Sheng , Wan Qing , Wang Miao
Published: March 2019

Objective:
Myocardial (M) ischemia/reperfusion (I/R) injury limits the efficacy of reperfusion therapy in patients with myocardial infarction (MI) and contributes to the development of heart failure. Nonsteroidal anti-inflammatory drugs (NSAIDs) that inhibit both cyclooxygenase (COX)-1 and COX-2, or those selective for COX-2 inhibition, are associated with an increased risk of heart failure. Although suppression of COX-2-derived prostaglandin (PG) I
2
predisposes cardiovascular risks, it remains unknown whether microsomal (m) PGE synthase (S)-1, a therapeutic target alternative to COX-2, might participate in MI/R injury.

Approach and Results:
Mice deficient in COX-1 or -2, mPGES-1, or endothelial PGE receptor-4 (EP4), together with pharmacological interventions, were utilized in a mouse model of MI/R. Microvascular perfusion was assessed in vivo using laser Doppler flow technique. COX-1 deficiency reduced biosynthesis of PGE
2
and increased infarct size in the heart after MI/R. Deletion of mPGES-1 also depressed PGE
2
while exacerbated MI/R injury. Cardiac perfusion was impaired by mPGES-1 deletion following reperfusion, without change in baseline flow. Consistently, mPGES-1 deletion abolished arteriolar dilation in I/R, and mPGES-1-derived PGE
2
acting on EP4 receptor restrains myeloid cell adhesion to endothelial cells in vitro and limits leukocyte adhesion to vasculature in I/R in vivo. Endothelium-restricted deletion of EP4 receptor impaired microcirculatory perfusion and exacerbated cardiac injury in MI/R. By contrast, treatment with misoprostol, a clinically available PGE
2
analogue, improved cardiac microcirculation post MI and protected against I/R injury.

Conclusions:
mPGES-1-derived PGE
2
and endothelial EP4 receptor protect against MI/R injury via preserving cardiac microcirculation. mPGES-1 inhibitors may harm microcirculation in patients with acute MI undergoing revascularization. Inhibition of COX-1-derived PGE
2
may contribute to the cardiovascular side effects of NSAIDs in the setting of I/R.

Key Words:
myocardial infarction, ischemia reperfusion, cyclooxygenase, prostaglandin E synthase-1, PGE
2
, EP4, microcirculation, endothelium
Abstract 278: Developmental Contractile Function Modulates Notch1b-Mediated Valvular Leaflet Development
Authors: Hsu Jeffrey J , Chen Junjie , Vedula Vijay , Chen Cynthia , Lee Juhyun , Tintut Yin , Demer Linda L , Marsden Alison , Hsiai Tzung K
Published: March 2019

Cardiac valve formation is a complex process affected by blood flow, but the mechanotransduction mechanisms underlying valvulogenesis remain incompletely understood. Using four-dimensional (4-D) light-sheet imaging, we evaluated the effects of pharmacological and genetic hemodynamic modulation on ventriculobulbar (VB) valve formation in the outflow tracts (OFT) of transgenic
Tg(fli1a:GFP)
zebrafish embryos. Treatment with isoproterenol increased heart rate and cardiac contractility, increased
Notch1b
activity in the OFT, and resulted in the development of hyperplastic VB valve leaflets. While metoprolol treatment reduced heart rate without affecting contractility, there were no significant differences in
Notch1b
expression in the OFT or valve morphology. Meanwhile, BDM treatment significantly reduced heart rate and contractility, reduced
Notch1b
expression in the OFT, and prevented the formation of normal VB valve leaflets. Similarly, no VB valve leaflets were seen in the
cloche
mutant or
Tnnt2a
MO-injected embryos. Additionally, increasing blood viscosity by micro-injection of embryos with
EPO
mRNA increased
Notch1b
activity in the OFT and led to hyperplastic VB valve leaflets, but decreasing blood viscosity by
gata1a
MO micro-injection did not have any significant effect. Further, activation of the Notch signaling pathway with micro-injection of
NICD
mRNA resulted in hyperplastic VB valve leaflets. By integrating advanced optics with zebrafish genetics at the interface of developmental cardiac mechanics, we provide mechanotransduction insights into cardiac valve development within the OFT.
Abstract 284: Lrp1 Regulates Smooth Muscle Contractility by Modulating Cytoskeletal Dynamics and Ca
2+
Signaling
Authors: Muratoglu Selen C , Au Dianaly T , Ying Zhekang , Hernandez-Ochoa Erick , Fondrie William E , Hampton Brian , Migliorini Mary , Galisteo Rebeca , Schneider Martin F , Daugherty Alan , Rateri Debra L , Strickland Dudley
Published: March 2019

Objective:
Mutations affecting proteins in the extracellular matrix (ECM), microfibrils, or vascular smooth muscle cells (VSMCs) that impact contractility can predispose individuals to thoracic aortic aneurysms. We reported previously that the low-density lipoprotein receptor-related protein 1 (LRP1) maintains vessel wall integrity, and smooth muscle LRP1-deficient (

smLRP1
-/-

) mice exhibited aortic dilatation. The current study focused on the descending thoracic aorta (DTA) and examined the role of LRP1 in VSMC contractility and its potential effect on the vascular ECM.

Approach and Results:
LRP1-deficient VSMCs exhibited a synthetic phenotype characterized by higher proliferation rates and an increase in synthetic organelles, mitochondria, multivesicular bodies, and macropinocytotic vesicles. LRP1-deficient VSMCs also displayed changes in their microfilament and actin structure that result in an inadequate interaction with the ECM. Quantitative proteomics identified proteins involved in actin polymerization and contraction that were downregulated significantly in the DTA of

smLRP1
-/-

mice. Further analysis by qRT-PCR revealed attenuated mRNA levels for α-1D adrenergic receptor (
adra1d
) and calcium voltage-gated channel subunit α1 C (
cacna1c
) in

smLRP1
-/-

aortas. Isometric contraction assays confirmed aberrant contraction of

smLRP1
-/-

aortic rings when stimulated with vasoconstrictors. Furthermore, intracellular calcium imaging identified defects in response to a ryanodine receptor agonist in

smLRP1
-/-

aortic rings.

Conclusions:
These results suggest that LRP1 is required for maintaining the VSMC contractile phenotype and identifies a novel role for LRP1 in calcium homeostasis that potentially protects against aneurysm development.
Abstract 300: The Vascular Ecto-enzyme, CD39 Protects from Venous Thrombogenesis by inhibiting Innate Immune Activation
Authors: Kanthi Yogendra , Jacobs Benjamin N , Chi Liguo , Yadav Vinita , Zhao Raymond , Banka Alison , Knight Jason S , Pinsky David J
Published: March 2019

Background:
Venous thrombosis (DVT) is a serious health concern, with growing incidence in an aging, comorbid population. DVT is marked by sterile inflammation and innate immune activation. Early participants in DVT include platelets, and neutrophils (PMN) which extrude their DNA to form thrombus-potentiating extracellular traps (NETs). To identify improved therapeutic targets at the nexus of inflammation and coagulation, we focused on the vascular ectonucleotidase CD39, found on leukocytes and the endothelium. CD39 dissipates extracellular ATP & ADP, thrombo-inflammatory “danger” signals. We hypothesized that CD39 is a critical enzyme in venous homeostasis, restraining unchecked inflammation and thrombosis in DVT.

Methods:
DVT was induced in
Cd39
+/-
and WT control mice using a “flow-restriction” model of inferior vena cava (IVC) stenosis. Thrombus frequency and size were assessed 48h after DVT induction. A novel flow cytometry method, immunoblot, immunostaining were used to examine thrombus cellular content, fibrin, NETs, and inflammasome activation.

Results:
Cd39
+/-
mice had a significantly higher thrombus frequency (2.5-fold) and clot size (3-fold), with more fibrin content by immunoblot. Flow cytometry revealed exaggerated PMN recruitment to the growing thrombus in
Cd39
+/-
mice, with more NETs
in vitro
, and within DVT compared with WT mice.
Cd39
+/-
mice also had amplified DVT inflammasome activity
in vivo
, measured by increased NFkB phosphorylation and mature IL-1 beta compared with WT DVT. Flow cytometry of the thrombus revealed an increase in activated platelet-PMN heteroaggregates within
Cd39
+/-
DVTs, indicating enhanced innate-coagulation system crosstalk.

Conclusion:
CD39 is a critical vasculoprotective ecto-enzyme in venous thrombogenesis.
Cd39
+/-
mice have increased DVT burden, fibrin deposition, activated platelet-PMN interactions, and exaggerated inflammasome activation compared with WT mice. By enrolling NET formation and inflammasome activation, CD39 posits a previously unexplored link between venous inflammation and thrombosis. Studies are underway to modulate inflammasome activation in
Cd39
+/-
mice, and to delineate the relative contributions of leukocyte/endothelial CD39 in venous thrombosis.
Abstract 313: Loss of Contractile Smooth Muscle Cell Phenotype in a Mouse Model of Marfan Syndrome
Authors: Gharraee Nazli , Lessner Susan M
Published: March 2019

Objectives:
Thoracic aortic aneurysm (TAA) is a chronic vascular disease and the 15th leading cause of death in the US. TAA can result from a genetic disorder such as Marfan syndrome (MFS). MFS patients typically have fibrillin-1 deficiency, causing elastic fiber defects in the aortic media that initiate a series of cellular events. We sought to determine the association of smooth muscle cell (SMC) conversion from contractile to synthetic phenotype with changes in extracellular matrix (ECM), medial cell density, and arterial biomechanics.

Methods:
A mouse model of MFS (Fbn1 C1039G/+, Het) was used to study progression of TAA and cellular events over a 3-12 month time course. Small animal ultrasound was used to image the ascending aorta and to measure diameters at several positions. Immunohistochemistry (IHC) was performed on proximal aorta sections with smooth muscle-myosin heavy chain (SM-MHC) and SM alpha actin as markers for contractile SMCs. Second harmonic generation (SHG) microscopy was used to quantitate aortic medial collagen.

Results:
Ultrasound data show that aortic diameter significantly increases with time in Het mice. There is a statistically significant difference between genotypes independent of time. IHC images demonstrate that while SM-MHC expression in wild type (WT) mice remains nearly constant, in Het mice at 3 months the expression is comparable to that of WT mice but decreases at 6 months and starts to recover to the WT level by 12 months. SHG images show a time dependent increase in medial collagen expression in Het mice vs. WT. These results suggest loss of contractile SMCs in the media and remodeling of ECM.

Conclusion:
Our results to date demonstrate loss of contractile SMCs and increased medial collagen deposition in the proximal aorta during TAA progression. Loss of contractile cells together with changes in ECM are expected to alter aortic biomechanics. To quantify these alterations, we are currently studying active and passive biomechanics of ascending aortas of WT and Het mice.