bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 157: Micromechanics of Blood Clots
Authors: Eng Wilson , Kim Max , Pham Stephanie , Saha Amit , Ramasubramanian Anand K , Lee Sang-Joon
Published: March 2019

Understanding clot biomechanics is critical for the treatment of cardiovascular diseases. Based on our recent observation that that the structural configuration of the clot network correlates well with the mechanical properties such as stiffness, we hypothesized that the heterogeneity in the mechanical response of the microstructure dictates clot micromechanics and hence the macroscopic behavior. To test this hypothesis, we have custom-developed a microextensometer device coupled to a microscope to probe and image microstructural changes and micromechanical behavior of fibrin and blood clots. 20 μL clots were pulled at a prescribed strain rate of 60 μm/s using a programmable nano-positioner, and the force was measured using a 10 g load cell and acquired at 500 Hz. From the stress-strain measurements, we observed that both FFP and blood clots showed non-linear and abrupt changes in resistive tensile force in response to constant strain rate (Fig. 1A). Using fiduciary markers, we observed that cross-linked, but not uncrosslinked, fibrin clots showed a microscopically non-uniform deformation in response to macroscopically constant strain rate. Further, computational analysis of the mechanical response of clot microstructure to an applied stress revealed heterogeneity in strain energy distribution dictated by the network properties (Fig. 1B). Together, our results suggest that the heterogeneity in microscale translates to the non-linear response at the macroscale, and will ultimately dictate the pathophysiology of thrombosis.
Abstract 159: Neurotrophin 3 Plays a Critical Role in TLR4-mediated Fibrogenic Response in Human Aortic Valve Interstitial Cells
Authors: Yao Qingzhou , Ao Lihua , Fullerton David A. , Yao Xianzhong
Published: March 2019

Aortic stenosis is a chronic inflammatory condition, and valvular fibrosis and calcification are significant pathological changes in the progression of aortic stenosis. Aortic valve interstitial cell (AVIC) proliferation and over-expression of extracellular matrix (ECM) proteins promote valvular fibrosis. Although pro-inflammatory stimuli are the culprits of aortic valve fibrosis, the mechanism by which they induce aortic valve fibrogenic change remains incompletely understood. Our recent findings suggest that AVICs from diseased aortic valves express higher levels of neurotrophin 3 (NT3) protein and that NT3 is capable of up-regulating AVIC proliferation and ECM protein expression. In this study, we tested the
hypothesis
that pro-inflammatory stimulation up-regulates NT3 production to enhance the pro-fibrogenic activity in human AVICs.

Methods and Results:
Human AVICs isolated from normal valves were treated with Toll-like receptor 4 (TLR4) agonist lipopolysaccharide (LPS, 0.20 μg/ml) for 3 to 28 days in the presence or absence of a neutralizing antibody against NT3. LPS elevated BrdU incorporation, collagen III expression and collagen deposition. Neutralization of NT3 markedly reduced these pro-fibrogenic changes. Interestingly, LPS up-regulated NT3 levels in human AVICs in a TLR4-dependent fashion, and inhibition of either Akt or ERK1/2 attenuated the effect of LPS on NT3 expression. Recombinant NT3 induced human AVIC proliferation, collagen III expression and collagen deposition through the Trk receptors. Inhibition of the Trk receptors abrogated the pro-fibrogenic effect of LPS on AVICs.

Conclusions:
Stimulation of TLR4 in human AVICs elevates the pro-fibrogenic activity through up-regulating the production of neural growth factor NT3. The Akt and ERK1/2 pathways are involved in TLR4-mediated NT3 up-regulation, and the Trk receptors mediate the pro-fibrogenic effect of TLR4 stimulation in human AVICs. These findings reveal a molecular mechanism underlying pro-inflammatory elevation of AVIC pro-fibrogenic activity and indicate the therapeutic potential of antagonizing NT3 for suppression of aortic valve fibrosis in an inflammatory setting.
Abstract 169: Lipoproteins and their Modified Forms Regulate Smooth Muscle Cell Calcification
Authors: Akers Emma J , Mulangala Jocelyne , Psaltis Peter J , Bursill Christina , Nicholls Stephen J , Di Bartolo Belinda A
Published: March 2019

Background:
Vascular calcification (VC), alongside atherogenic lipoprotein profiles, have been correlated with poor cardiovascular outcome, however there is a paucity of literature exploring the relationship between the two regarding VC progression. We therefore aim to examine the roles of lipoprotein species and their oxidised forms on both medial and intimal VC.

Methods:
Human aortic smooth muscle cells (HAoSMC) were pre-treated with 200 μg/ml high (HDL), low (LDL) and very low (VLDL) density lipoproteins for 24 hours before treating cells with a calcification medium (CM; Ca 2.7mM, PO
4
2.0 mM). Cells were harvested using an alizarin red (ARS) calcification assay, or at various time points for qPCR analysis. In parallel,
in vivo
studies using apolipoprotein E knock-out mice were fed an atherogenic diet for 40 weeks and received reconstituted HDL (rHDL) infusions containing apoA-I (20mg/kg) and 1-palmitoyl-2-linoleoyl phosphatidylcholine during the final 4 weeks of the study. Tissues were harvested and stained for plaque assessment (H&E) and calcium deposits (ARS).

Results:
Pre-treatment of HAoSMC with rHDL inhibited calcification (43.9%, p<0.001), whereas ox-rHDL removed its protective effects. Likewise, ox-LDL (77.1%, p<0.05) also upregulated calcium deposition and interestingly ox-VLDL significantly decreased calcification (70.5%, p<0.05) with their native counterparts having no effects. PCR measures of calcification markers Runx2, RANKL and alkaline phosphatase show a time-dependent increase in expression as calcification occurs. In animal studies, no change in weight gain, cholesterol or triglyceride levels were observed with treatment. In addition, rHDL infusions did not alter plaque size however ARS staining of the brachiocephalic artery demonstrated a significant reduction (6.58%, p<0.05) in calcification present in the atherosclerotic plaque.

Conclusions:
This study is the first to demonstrate the effects of lipoproteins on VC
in vitro
and the effects of rHDL on
in vivo
VC. Somewhat in accordance to the roles of lipoproteins in atherosclerosis, HDL and ox-VLDL show a reduction of calcification, where-as ox-LDL enhances calcification of HAoSMC.
Abstract 171: Direct versus Calculated LDL Cholesterol and C Reactive Protein in Cardiovascular Disease Risk Assessment in the Framingham Offspring Study
Authors: Ikezaki Hiroaki , Fisher Virginia A , Liu Ching-ti , Cupples L. Adrienne , Nakajima Katsuyuki , Asztalos Bela F , Furusyo Norihiro , Hayashi Jun , Schaefer Ernst J
Published: March 2019

Background:
Elevated serum LDL cholesterol (LDL-C) and high sensitivity C reactive protein (hsCRP) levels have been identified as major risk factors for cardiovascular disease (CVD). Our goal was to assess direct versus calculated LDL-C and hsCRP levels as compared to standard risk factors in the prospective Framingham Offspring Study.

Methods:
Stored frozen plasma samples (-80 degrees C) obtained after an overnight fast from male and female participants free of CVD at cycle 6 of the Framingham Offspring Study were used (n=3,147, mean age 58 years) and 677 or 21.5% developed a CVD endpoint over 16 years. Total cholesterol, triglycerides, HDL cholesterol (HDL-C), direct LDL-C, and hsCRP were measured by standardized automated analysis, and LDL-C was also calculated.

Results:
For inclusive CVD risk on univariate analysis significant factors in order included the standard risk factors age, hypertension, HDL-C, hypertension treatment, gender, diabetes, smoking, and total cholesterol, as well as the non-standard risk factors non-HDL-C, direct LDL-C, calculated LDL-C, triglycerides (TG), and hsCRP. On multivariate analysis only direct LDL-C and hsCRP were still significant after inclusion of the standard model. Both parameters significantly improved the model C statistic and the net risk reclassification index. The same findings were noted for other CVD risk categories including hard CVD with procedures and hard CVD.

Conclusions:
Our data indicate that direct LDL-C is superior to calculated LDL-C in CVD risk prediction, and that both direct LDL-C and hsCRP add significant information to CVD risk prediction versus the standard model.
Abstract 173: Sodium Salicylate Modulates Reverse Cholesterol Transport and HDL Proteome within Obesity
Authors: Kajani Sarina , O'Reilly Marcella , Guo Weili , Dillon Eugene , McGillicuddy Fiona C
Published: March 2019

Obesity is associated with increased risk of cardiovascular disease (CVD) due to imbalances in lipid metabolism and leads to maladaptive inflammatory responses. This study assessed the effects of an obesogenic diet supplemented with anti-inflammatory sodium salicylate (NaS) on hepatic health, HDL proteomic quality, HDL functionality and Reverse Cholesterol Transport (RCT).

RCT was assessed after 24 weeks in C57BL/6 mice fed a high fat (HFD) (60% fat) ± NaS (6g/kg) diet or low-fat diet (LFD, 10% fat) by tracing
3
H-cholesterol movement from labelled macrophages, injected intraperitoneally, into plasma, liver and fecal compartments. HDL particles were separated from plasma by fast protein liquid chromatography (FPLC) and associated proteins were identified by mass spectrometry. HDL particles were incubated with
3
H-cholesterol-labelled macrophages
ex vivo
and cholesterol efflux to HDL particles was determined by liquid scintillation counting (LSC).

Both HFDs significantly increased HDL cholesterol mass. Nonetheless, HFD alone impaired hepatic movement of
3
H-cholesterol to fecal compartments coincident with increased hepatic lipid infiltration and inflammation. NaS supplementation increased all steps of macrophage-to-feces RCT with partial preservation of hepatic transporters while impeding hepatic lipid load and reducing markers of inflammation (ALT & AST). HFD resulted in enrichment of HDL particles with coagulant proteins (fibrinogen, coagulation factor IX) and a dissociation of anti-thrombin and angiotensinogen compared to HFD+NaS. NaS supplementation also preserved ABCA1-mediated efflux capacity of HDL particles.

Obesogenic diets augmented with NaS resulted in profound differences within the RCT pathway and HDL proteome. HFD supplemented with NaS was associated with improved hepatic condition and HDL proteomic profile with an increase of anticoagulant and anti-atherogenic proteins on HDL despite development of obesity.
Abstract 175: Dynamin-Related Protein 1 Regulates Proteostasis and Proprotein Convertase Subtilisin/Kexin Type 9 Secretion
Authors: Rogers Maximillian A , Hutcheson Joshua D , Goettsch Claudia , Halu Arda , Singh Sasha , Higashi Hideyuki , Lee Lang H , Wang Lixiang , Schlotter Florian , Morgan Stephanie , Okui Takehito , Yamazaki Yukiyoshi , Daugherty Alan , Nomura Masatoshi , Aikawa Masanori , Aikawa Elena
Published: March 2019

Objective:
Dysfunctional protein homeostasis (proteostasis) contributes to cardiovascular and metabolic disorders. We and others associated the mitochondrial fission protein, Dynamin-related protein 1 (DRP1) with cardiometabolic disease. Liver DRP1-deficiency reduces serum lipids and very-low density lipoprotein secretion in high-fat fed mice; whether DRP1 mediates these effects via proteostasis regulation is unknown.

Approach and Results:
Using mass spectrometry integrated with network analysis to map the human liver secretome, we found DRP1 associated with cardiovascular disease modules and lipid pathways. Electron microscopy revealed human liver DRP1 at mitochondria, cytosol, vesicles, endoplasmic reticulum (ER), and clustered at membrane tethered to ER exit sites. DRP1 small molecule inhibition (Mdivi-1) or CRISPR/Cas9-mediated
DRP1
deletion in human liver cells, and
Drp1
-liver deficiency in mice reduced autophagic flux without impairing the amino acid metabolome, or activating the autophagy inhibitor, mammalian target of rapamycin complex 1. DRP1 partially co-localized and co-immunoprecipitated with the ER trafficking and autophagy regulator, Syntaxin 17, in human liver tissue and cells. DRP1 inhibition reduced Proprotein convertase subtilisin/kexin type 9 (PCSK9) secretion in human liver cells and mice (-78.5%), and altered trafficking of the PCSK9-binding and ER maintenance chaperone, Glucose-regulated protein 94. Co-treating human liver cells with Mdivi-1 and proteasome inhibitor (MG132), non-transcriptionally increased intracellular PCSK9, while maintaining Mdivi-1-mediated reduced PCSK9 secretion.

Conclusions:
We propose a novel function of DRP1 in the regulation of proteostasis, wherein DRP1 may cluster, then tether and/or constrict nascent autophagy-associated membrane at the ER via its interaction with Syntaxin 17. DRP1 inhibition likely reduces lipoprotein and PCSK9 secretion in part by impairing autophagic flux leading to compensatory chaperone-mediated proteasomal degradation for ER maintenance. Proteostasis regulation and the cellular function of DRP1 is more complex than previously thought, potentially providing new avenues to therapeutically target cardiometabolic disease.
Abstract 177: Glucagon Receptor Signaling-Mediated Regulation of PCSK9 and Cholesterol Metabolism
Authors: Spolitu Stefano , Ozcan Lale
Published: March 2019

Glucagon is one of the key hormones important in hepatic glucose homeostasis, and the pathophysiological role of hyperglucagonemia and unopposed hepatic glucagon action in type 2 diabetes (T2D) is now well-established. Accordingly, there has been great interest in developing glucagon receptor antagonists (GRAs) as a treatment for T2D. Although phase 2 clinical trials have shown that GRAs effectively lower blood glucose in T2D subjects, they increase plasma low density lipoprotein cholesterol (LDL-C), which has presented a significant block to their development. Consistent with a role of glucagon in cholesterol metabolism, recent studies suggested that proprotein convertase subtilisin/kexin type 9 (PCSK9), which increases plasma LDL-C through targeting LDL receptor (LDLR) for degradation, can be regulated by fasting, however, in-depth mechanistic information is lacking. In order to test the functional importance of hepatic glucagon action on cholesterol metabolism, we silenced hepatic glucagon receptor (GcgR) in obese mice using AAV8-mediated shRNA treatment. Consistent with previous reports, this treatment effectively lowered blood glucose in obese mice without a change in body weight. Moreover, GcgR silencing, like GRAs in humans, significantly increased plasma LDL-C. In search for the mechanism, we found that inhibition of GcgR significantly lowered hepatic LDLR protein levels and increased both hepatic and circulating PCSK9, without an effect on cholesterol synthesis. To determine causation, we silenced hepatic GcgR together with PCSK9 using AAV8 and found that this intervention restored hepatic LDLR and prevented the increase in plasma LDL-C. Further mechanistic studies showed that GcgR silencing in hepatocytes did not increase
Pcsk9
mRNA. Rather, blocking GcgR increased the half-life of PCSK9 protein by suppressing signalling through exchange protein activated by cAMP-2 (Epac2). In particular, the ability of GcgR silencing to increase PCSK9 and suppress LDLR protein levels was mimicked by hepatocytes lacking Epac2. Thus, GcgR signalling through Epac2 appears to have critical effects on processes that regulate cholesterol metabolism through PCSK9.
Abstract 186: Deletion of Ppard in Cd11c+ Cells Attenuates Atherosclerosis in Apoe Knockout Mice
Authors: Tian Danyang , Wu Yalan , Chawla Ajay , Huang Yu , Tian Xiao Yu
Published: March 2019

Atherosclerosis is a chronic sterile inflammation of the vascular wall triggered by hyperlipidemia. The role of dendritic cells (DCs) in the development of atherosclerosis has not been recognised until the last decade. DCs can engulf lipids to adopt a foam cell-like appearance that may constitute the earliest stages of plaque formation. DCs may also recruit T cells to the inflamed vessel wall via secretion of chemokines and stimulate T cell responses via cytokines. PPARD is a nuclear receptor, which acts as a sensor of native and oxidized fatty acids. We examined whether PPARD regulates DC function in response to hyperlipidemia and affect atherosclerotic lesion growth. We used Ppard
fl/fl
mice and Itgax-cre (CD11c-Cre) mice crossed with Apoe
-/-
mice to generate Ppard
f/f
;CD11c
Cre/+
;Apoe
-/-
as DC-specific knockout of PPARD on Apoe-KO background (Ppard
DC-KO
) and Ppard
f/f
;Apoe
-/-
(Ppard
DC-WT
) as controls. Ppard
DC-KO
and Ppard
DC-WT
were fed with high cholesterol diet for 4 months. Tissues were dissected and digested to obtain single cell suspension for flow cytometric analysis. Bone marrow derived DCs were isolated and cultured in RPMI with serum and GM-CSF and maturated with LPS.Atherosclerotic lesion size and collagen deposition decreased in Ppard
DC-KO
mice. Less CD4 and CD8 T lymphocytes were found in the atherosclerotic lesion of Ppard
DC-KO
mice comparing to Ppard
DC-WT
mice. Ppard deletion in DCs reduced DC infiltration especially CD11b
+
CD103
-
DCs in the atherosclerotic lesion. Production of IFNγ from CD4
+
T cells decreased in Ppard
DC-KO
mice. In BMDCs from Ppard
DC-KO
mice, LPS and palmitic acid induced expression of co-stimulatory molecules CD80 and CD86, as well as TNF were decreased. LDL uptake was attenuated in BMDCs from PpardDC-KO mice. Our results suggested that PPARD may be involved in DC mediated T cell activation in response to hyperlipidemia. Deletion of PPARD in DCs attenuated plaque formation in atherosclerosis. Whether lipid sensing and uptake by PPARD is required for the enhanced inflammatory response in atherosclerotic mice requires further study. (This study is supported by Hong Kong Health Bureau HMRF 05162906 and 01150057, Hong Kong RGC T402/13-N and CRF C4024-16W, and CUHK Direct grants)
Abstract 189: Novel Drug Development Controlling Residual Inflammatory Risk in Diabetic Atherosclerotic Disease
Authors: Yussman Martin , Wagner David , Vaitaitis Gisela , Waid Dan
Published: March 2019

Sustained inflammation is a crucial pathologic component in atherosclerosis and type 2 diabetes (T2D). A common molecular player driving auto-inflammation in both diseases is the CD40/CD154 inflammatory dyad. By normalizing the aberrant contact dependent interaction of the CD40/CD154 dyad, and resultant auto-inflammation, multiple studies have shown both prevention and therapeutic efficacy in both diseases.

We designed a series of peptides derived from the CD154 protein sequence that are capable of binding directly to the CD40 receptor to interrupt the inflammatory signal pathways. Two peptides, KGYY6 and KGYY15, were highly effective, initially demonstrating efficacy in type one diabetes mouse models. KGYY6 was chosen for additional testing in the T2D model with atherosclerosis.

ApoE-/- mice were utilized due to their ability to develop severe vascular disease and acquire the elements of T2D after 16 weeks of a high fat diet (HFD). KGYY6 was administered by IV tail injection at a dose of 1mg/kg and compared to controls given vehicle only.

Aortic en-Face analysis with Sudan IV stain demonstrated significant reduction in plaque in KGYY6 treated mice. Decreases in plaque area and changes in both smooth muscle and collagen measurement were additionally noted by sequential 5um aortic cross sections from the aortic valve leaflets into the ascending aorta. In-vitro analysis of CD3+CD4+CD40+ splenic cells demonstrated a reduction in inflammatory cytokine expression in response to KGYY6 treatment, specifically IL2, IFNγ, and IL17, which are potent cytokines in atherosclerosis.

Western blot analysis performed on adipose and muscle tissue demonstrated an increased expression of the glucose transport protein GLUT 4, with corresponding glucose tolerance testing demonstrating increased glucose tolerance and improved insulin sensitivity with lowered plasma insulin level.

KGYY6 normalizes the aberrant CD40/CD154 interaction, reducing inflammatory cytokines and regulating glucose, all together resulting in abrogating atherosclerosis.
Abstract 204: Diabetes Influence Infragenicular Arterial Segment Phospholipid Profiles
Authors: Yang Chao , Hsu Fong-Fu , Yan Yan , Darwesh Malik , Zayed Mohamed
Published: March 2019

Diabetes patient have higher risk of PAD. Our group previously demonstrated that CEA plaque lipidomics are altered by diabetes. It remains unknown whether similar alterations also occur in infra-inguinal PAD. Between 2015 to 2017, patients undergoing major lower extremity amputation were recruited into a vascular surgery tissue biobank. At the time of amputation, infra-inguinal arterial segment with maximally (Max) and minimally (Min) disease were harvested. lipids were extracted using a modified Bligh-Dyer technique and evaluated by Electrospray-ionization tandem mass spectrometry. The relative proportions of all major phospholipid species (including phosphatidylcholine, PC; ceramide, Cer; phosphatidylethanolamine, PE; phosphatidylinositol, PI; and phsophatidylserine, PS) were derived. Paired one-sample sighed rank test was used to evaluate the differences between Max and Min diseased arterial segments, and Wilcoxon two-sample test was used between diabetic and non-diabetic group. 14 diabetic (DM) and 7 non-diabetic(NDM) patients were enrolled with well-matched demographics. Analysis of the major phospholipid families, there were no differences between DM and NDM in Max segments. However in Min segments there were differences (58% lower PI in DM patients, P<0.03). Further sub-group analysis was performed at max and min in DM and NDM separately and found differences in PCs, aPCs, PEs, pPEs, Cers. The most highly differentially expressed lipids included PC 40:3(76%, P=0.01), PC 40:5(60%,P=0.04 ), aPC38:3 (83%, P=0.01), aPC38:2(79%, P=0.04) ,and these have been shown previously to be important in arachidonic acid mediated inflammation. Max and Min diseased segments from infra-ingunial arterial segments from diabetic and non-diabetic patients demonstrate significantly different lipid contents. These findings highlight the profound biochemical differences in plaque phospholipid biology in the diabetes and provide a platform for future exploration in diseased oriented treatment strategies.
Abstract 213: Human Genetic Variations in Neuroimmune Guidance Cues and their Role in Premature Atherosclerosis
Authors: Bruikman Caroline S , Vreeken Dianne , van Capelleveen Julian C , Zhang Huayu , Pinto-Sietsma Sara J , Dallinga-Thie Geesje M , Dallinga-Thie Geesje M , Hovingh G. K , van Gils Janine M
Published: March 2019

Introduction:
Neuroimmune guidance cues (NGCs), a family of proteins originally known for controlling neuronal migration, have been shown to be involved in atherosclerosis in mice models by regulating adhesion of monocytes to the vascular endothelium. The NGC family consists of multiple receptors and ligands enabling an interplay between inflammatory cells and the vasculature endothelium. We set out to investigate the role of NGCs in atherosclerosis in humans.

Methods and results:
We sequenced a total of 77 NGC genes in a unique cohort of 89 patients with premature atherosclerotic (PAS), in whom no clear CVD risk factor was present. A total of 178 rare variants were identified and we classified the pathogenicity of these rare (MAF<0.05) variants by high Combined Annotation Dependent Depletion (CADD) scores. Based on literature and cosegregating pedigrees of the 88 possible deleterious variants we chose two variants in Netrin1 (NTN1) and EphrinB2 (EFNB2) to explore and validate their causality in human atherosclerosis. The expression of both these genes is regulated by flow and inflammatory stimuli. Under atherosclerotic conditions endothelial cells upregulate EFNB2 while NTN1 is downregulated. In addition, using immunohistochemical staining on human aorta’s, we found that NTN1, which is normally expressed on human endothelial cells, is less abundant in increasing stages of atherosclerosis. For EFNB2, a cell surface transmembrane ligand for Eph receptors, we performed several functional assays using HUVEC cells that overexpress EFNB2 , showing that EFNB2 is affecting endothelial cell barrier function, as well as proliferation.

Conclusion:
In patients with PAS rare missense variants in NGC genes were identified. Further functional- and expression assays showed an effect of NTN1 and EFNB2 on different aspects of the human atherosclerotic process. The results obtained from these studies substantiate the role of NGCs in human atherosclerotic disease and may further identify NGCs as novel potential therapeutic targets in our strive against cardiovascular disease.
Abstract 217: Coronary Artery Disease Locus 1p32.2 Harbors a Flow-Sensitive Endothelial Enhancer that Regulates PLPP3
Authors: Fang Yun , Krause Matthew , Civelek Mete , Romanoski Casey
Published: March 2019

Genome-wide association studies (GWAS) have identified chromosome 1p32.2 as one of the loci most strongly associated with coronary artery disease (CAD) susceptibility; however, the causal mechanism related to this CAD locus remains poorly understood. A unique feature of atherosclerotic vascular disease such as CAD is that atherosclerosis develops preferentially at arterial sites of curvature, branching, and bifurcation where endothelial cells are activated by disturbed blood flow. Recent investigations by us and others strongly implicate PhosphoLipid PhosPhatase 3 (PLPP3, also known as PhosPhatidic-Acid-Phosphatase-type-2B/ PPAP2B or Lipid Phosphate Phosphohydrolase/LPP3) as the causal gene at this locus. PLPP3 encodes an enzyme that suppresses endothelial inflammation and promotes monolayer integrity by hydrolyzing the bioactive lipid lysophosphatidic acid (LPA). Our studies demonstrated that PLPP3 is significantly reduced in vascular endothelium exposed to disturbed flow in vitro and in vivo, as the result of increased miR-92a and reduced KLF2, when compared to cells subjected to unidirectional blood flow. In addition, CAD risk allele at rs17114036 located in 1p32.2 locus is associated with reduced PLPP3 expression in an endothelium-specific manner, shown by expression quantitative trait locus (eQTL). Employing Transposase-Accessible Chromatin using Sequencing (ATAC-Seq), ChIP-Seq, CRISPR/Cas9-based genome editing, luciferase reporters, and allelic imbalance assays, we report here that CAD SNP rs17114036 is located in an endothelial enhancer that is dynamically activated by athero-protective unidirectional flow and deletion of this enhancer causatively reduces PLPP3 expression in vascular endothelium. In addition, CAD risk allele at rs17114036 is associated with reduced activity of this enhancer and its response to unidirectional flow. These results described a new molecular mechanism by which human genetic variance and mechano-transduction converge on critical vascular functions related to atherosclerosis. Moreover, we have engineered innovative polymeric nano-carriers that preferentially target inflamed endothelium and deliver therapeutic nucleotides that intervene in miR92a-PLPP3 signaling in animal models of atherosclerosis.
Abstract 219: Role of Transcription Co-Factor Friend of GATA 2 (FOG2) in a Hypertensive-Diabetic Mouse Model of Coronary Microvascular Disease
Authors: Guerraty Marie A , Szapary Hannah J , Berrido Andrea , Arany Zoltan P , Rader Daniel J
Published: March 2019

Coronary microvascular disease (CMVD), or disease of the coronary pre-arterioles, arterioles, and capillaries, has become an increasingly well-recognized cardiac pathology. Characterized by microvascular remodeling, wall thickening, lumen obliteration, endothelial dysfunction, capillary rarefaction, and perivascular immune cell infiltration, CMVD can lead to inadequate myocardial perfusion, angina, heart failure, or myocardial infarction. Despite significant clinical ramifications, our understanding of the pathophysiology of CMVD remains nascent. Friend of GATA 2 (FOG2, also ZFPM2) is a crucial transcriptional co-factor in coronary development; and cardiomyocyte expression of FOG2 is required for the maintenance of the coronary microvasculature in adult mice. We hypothesize that FOG2 is activated in CMVD, and that Hypoxia-Inducible Factor 1a (HIF1a) may play a role in FOG2 activation. To test this hypothesis, we developed a hypertensive-diabetic (HTN/DM) mouse model of CMVD. Briefly, adult female C57BL/6 mice were treated with Angiotensin II (1mg/kg/day osmotic pump for 8 weeks) and low-dose streptozotocin (35 mg/kg IP for 4 days) (n=5) or saline (n=4). HTN/DM mice had increased systolic blood pressure (142±2 mmHg v 109±1 mmHg, p<1e4), mild fasting hyperglycemia (182±9 mg/dl v 149±10 mg/dl, p=0.04), and decreased glucose tolerance (p<3e4). These resulted in decreased cardiac capillary density (149±10 v 164±4 phf, p=0.008) and evidence of microvascular pathology including vessel wall thickening and immune cell infiltration. HTN/DM mice had mild LV hypertrophy, preserved EF, and diminished coronary hyperemic velocities by echocardiography. Cardiac FOG2 protein was increased in HTN/DM mice (p<0.05). To examine the relationship between HIF and FOG2
in vitro
, AC16 cardiomyocytes were treated with HIF-hydroxylase inhibitor DMOG (0.1mM, 1mM) for 16 hours. There was a dose-dependent increase in FOG2 expression and nuclear translocation. In summary, we developed a mouse model of CMVD with mild hypertensive-diabetic insults which are relevant to human disease. We found that FOG2 protein is increased in CMVD suggesting that FOG2 may be relevant to adult CMVD pathophysiology
in vivo
. We additionally provide
in vitro
evidence that FOG2 is induced in HIF-activating conditions.
Abstract 226: Proprotein Convertase Subtisilin Kexin Type 9 Knockout Mice are Protected from Valvular Calcification
Authors: Poggio Paolo , Songia Paola , Chiesa Mattia , Barbieri Silvia , Cavallotti Laura , Valerio Vincenza , Ferri Nicola , Zanotti Ilaria , Camera Marina
Published: March 2019

Background:
Aortic valve calcification (AVC) is one of the most common form of heart valve disease and affects 3% of the population. No pharmacological treatments have been identified yet; however, proprotein convertase subtisilin kexin type 9 (PCSK9) inhibitors have been proposed to reduce AVC progression. We aimed to evaluate the role of PCSK9 in the aortic valve calcification.

Methods:
We use PCSK9
-/-
and PCSK9
+/+
(WT) mice littermates (n=3-4 per group, 12 month-old female, normal diet). When aortic valves were excised, total calcium content was measured and valve interstitial cells (VIC) were isolated. Calcification assays were performed on aortic VICs, left untreated or treated with β-glycerophosphate and ascorbic acid (βGAA) for seven days on plastic dishes. MinION RNA-Seq was used to evaluate the differential expression patterns in the two groups.

Results:
Total calcium content of aortic valve from PCSK9
-/-
mice was significantly lower than WT (1.5±0.7
vs
10.1±0.5 ng of calcium/μg of total protein, respectively; p=0.0002). Calcium quantification revealed that PCSK9
-/-
VICs (untreated) were able to calcify at a less extent than WT (4.4±0.6
vs
8.0±0.5 ng/μg, respectively; p=0.0001). The βGAA treatment induced calcification in both cell types; however, a significant (p=0.01) lower calcification rate was noted in PCSK9
-/-
VICs (41.9±3.6 ng/μg) compared to WT (55.9±3.5 ng/μg). RNA-Seq of VICs from WT and PCSK9
-/-
mice revealed that 363 genes were differentially expressed more than two-fold (adjusted p value<0.05). Functional analysis showed that in PCSK9
-/-
VICs upregulated genes coordinate p38 cascade and cytoskeleton modifications, while downregulated ones control apoptotic signalling pathway, cell proliferation and adhesion, and oxidative stress.

Conclusion:
These preliminary results show for the first time that PCSK9 could play a direct role in AVC and thus, therapies against PCSK9 may be beneficial to patients affected by calcific aortic valve disease.
Abstract 227: Investigating the Role of Fatty Acid Binding Protein-4 in ApoE-/- Mice Aorta During Prolonged Percutaneous Retro-orbital Injections
Authors: Prathipati Priyanka , Walton Brian , Rodriquez-Aguayo Cristian , Sood Anil , Berestein-Lopez Gabriel
Published: March 2019

Background:
Atherosclerosis is a chronic inflammatory disease which is one of the major causes of stroke.Fatty acid binding protein-4 (FABP4) is a lipid chaperon that plays an important role in atherogenesis. It is mostly expressed in macrophages and adipocytes and acts as a mediator of inflammation. Atherosclerosis is a chronic inflammatory disease which is one of the major causes of stroke. We hypothesize that silencing FABP4 could be a novel therapeutic treatment for atherosclerosis, hence a liposomal small interfering RNA (siRNA) delivery system to silence FABP4 was developed.

Materials and methods:
Under an appproved IACUC protocol, male

ApoE
-/-

mice (N=8) were fed high fat diet from 6 weeks of age until 12-16 weeks. At 11 weeks of age, the mice were injected with 0.1ml (500μg/ml) of FABP4/control siRNA liposomes tagged with fluorescent Qdot (QD) 625. The injections were administered through percutaneous retro-orbital route twice a week for four consecutive weeks. Starting in the 12
th
week, two mice from each group were sacrificed every week. Aortas and serum were collected at the time of sacrifice. Aortic arch was dissected, stored in formalin and processed for paraffin sections. The rest of aorta was flash frozen for protein analysis. Protein levels of FABP4 in aortas will be analyzed by western blot and immunohistochemistry to investigate the silencing effect of FABP4 siRNA liposomes.

Results:
ApoE-/- mice aortic tissue sections showed the successful uptake of FABP4 siRNA liposomes.

Conclusion:
Percutaneous retro-orbital injections of FABP4 siRNA liposomes for four consecutive weeks might help to silence the FABP4 to greater extent in atherosclerotic model of mice.
Abstract 235: Ionizing Radiation Induces Endothelial Inflammation and Apoptosis via P90rsk-mediated Erk5 S496 Phosphorylation
Authors: Vu Hang T , Kotla Sivareddy , Ko Kyung Ae , Fujii Yuka , Tao Yunting , Medina Jan , Thomas Tamlyn , Hada Megumi , Sood Anil K. , Singh Pankaj Kumar , Milgrom Sarah A. , Krishnan Sunil , Fujiwara Keigi , Le Nhat-Tu , Abe Jun-ichi
Published: March 2019

Background:
Adverse cardiovascular events are a leading nonmalignant cause of morbidity and mortality among cancer survivors who have been exposed to ionizing radiation (IR), but the exact mechanism of the cardiovascular complications induced by IR remains unclear. In this study we investigated the potential role of the p90RSK-ERK5 module in regulating IR-induced endothelial cell inflammation and apoptosis.

Methods and Results:
Whole body radiation of mice with 2 Gy γ-ray significantly increased endothelial VCAM-1 expression; especially in the disturbed flow area
in vivo
. In vitro studies showed that IR increased p90RSK activation as well as subsequent ERK5 S496 phosphorylation in cultured human endothelial cells (ECs). A specific p90RSK inhibitor, FMK-MEA, significantly inhibited both p90RSK activation and ERK5 S496 phosphorylation, but it had no effect on IR-induced ERK5 TEY motif phosphorylation, suggesting that p90RSK regulates ERK5 transcriptional activity, but not its kinase activity. In fact, we found that IR-induced NF-kB activation and VCAM-1 expression in ECs were significantly inhibited by the over-expression of S496 phosphorylation site mutant of ERK5 (ERK5 S496A) compared to overexpression of wild type ERK5. Furthermore, when ECs were exposed to IR, the number of annexin V positive cells increased, and overexpression of ERK5 S496A, but not wild type ERK5, significantly inhibited this increase.

Conclusions:
Our results demonstrate that IR augmented disturbed flow-induced VCAM-1 expression in vivo. Endothelial p90RSK was robustly activated by IR and subsequently up-regulated ERK5 S496 phosphorylation, inflammation, and apoptosis in ECs. The EC p90RSK-ERK5 signaling axis can be a good target to prevent cardiovascular events after radiation therapy in cancer patients.
Abstract 243: Familial Hypercholesterolemia Screening at a Large Corporate Clinic in Japan
Authors: Hara Masumi , Iso-O Naoyuki , Sugiura Anna , Takai Atsuko , Takeuchi Hideyuki , Hara Sonoka , Yamada Takaharu , Ito Seigo , Tsukamoto Kazuhisa
Published: March 2019

Background and Aims:
Familial hypercholesterolemia (FH) is a high risk of developing premature coronary artery disease even in heterozygotes. It is reported that the diagnostic rate in Japan and the United States is extremely low compared with the Netherlands and a few countries. The diagnostic criteria of FH vary from country to country. Using the JASb(Japan Atherosclerosis Society) diagnostic criteria, it has been shown that sensitivity and specificity in patients who visited lipid clinics are high, but those in the general population are not clear. In this study, we tried to screen FH in a clinic of a large Japanese corporation.

Methods:
During the period from July 1, 2014 to October 31, 2017, we extracted patients suspected of FH with LDL-C≧180mg/dL or with a family history of CAD at annual and follow-up medical checkup. In the extracted patients, family history of premature CAD and Achilles tendon thickening were evaluated. For patients to which additional information was added, FH diagnosis by JAS diagnostic criteria and by Dutch criteria were performed.

Results:
A total of 3,3136 medical checkups were set for about 6,300 subjects, and a total of 2,8108 visits were made (the examination rate was 84.8%). Among them, 328 patients with suspected FH were extracted due to LDL-C of 180 mg/dL or more, or family history of CAD. Among subjects additional information was obtained as of November 2017, 43 (19.6%) were diagnosed as FH on JAS criteria, and the number was higher than predicted from the estimated prevalence of FH. On the other hand, there were 24 patients who were "Definite" or "Probably" in Dutch criteria and 211 patients with LDL-C≧190 mg/dL. LDL-C of patients diagnosed as FH by JAS diagnostic criteria was 206.6 (45.3) mg/dL, while patients who did not meet the criteria also had high LDL-C values as 201.4 (18.6) mg/dL. 26.8% of patients who met the diagnostic criteria of FH had not received statin at the time of evaluation.

Conclusion:
The number of patients satisfying the JAS criteria was more than predicted from the prevalence, suggesting the possibility of over-diagnosis. On the other hand, there were many patients who do not satisfy the diagnostic criteria even when LDL-C is very high.
Abstract 245: Phenotype Discovery in Cardiovascular Patients Using Unsupervised Learning
Authors: Ross Elsie , Shah Nigam , Leeper Nicholas
Published: March 2019

Introduction:
Customer segmentation, as utilized in the retail industry, has direct applications to population health. By identifying unique patient subgroups, population health practitioners can produce health campaigns that are more effectively targeted. We describe a technique for identifying unique subgroups of patients with cardiovascular disease using unsupervised machine learning.

Methods:
Data were derived from a prospective, observational study of 1,329 patients who presented for elective, non-emergent coronary angiography at two tertiary care centers between 2004 and 2008 and were found to have CAD. Patients were measured along a total of 161 variables including demographic, clinical, physical activity, imaging, genomic and socioeconomic characteristics. We utilized a new technique called generalized low rank modeling (GLRM) coupled with K-means clustering to identify different patient subgroups. We then compared MACCE and mortality rates across clusters.

Results:
Utilizing GLRM we were able to identify 3 unique clusters of patients. While the rate of MACCE differed significantly across all 3 clusters (Figure 1), cluster 3, which represented the sickest group of patients, had significantly worse mortality rates than clusters 1 and 2. Clusters were distinguished by their prevalence of co-morbidites such as peripheral artery disease and diabetes, as well as engagement in physical activity, educational attainment, family history and genomic marker expression.

Conclusions:
Patients with cardiovascular disease are highly heterogeneous with different clinical, genomic and lifestyle traits. Unsupervised learning algorithms can be used to automate identification of important subpopulations using a wide range of data, above and beyond traditional risk factors. Identifying these unique groups of can help health practitioners better target health information and treatment strategies.
Abstract 261: Proteomic Analysis of Infrarenal and Suprarenal Aorta in a Kawasaki Disease Vasculitis Murine Model Associated with Infrarenal Abdominal Aorta Dilatation and Aneurysms
Authors: Noval Rivas Magali , Porritt Rebecca A , Parker Sarah J , Arditi Moshe
Published: March 2019

Background:
Kawasaki Disease (KD) is a childhood vasculitis that leads to coronary artery aneurysms and is the leading cause of acquired heart diseases among children in the USA. We have reported that the

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xtract (LCWE)-induced murine model of KD vasculitis and coronary arteritis also induces abdominal aorta dilatation and aneurysms (AAA) that involve exclusively the infrarenal (IR) region of the abdominal aorta (AA) without affecting the suprarenal (SR)-AA. AAA predisposes tissue to dissection and rupture, and is potentially life-threatening in humans. While differences in blood flow, tissue elasticity and different embryonic origin of vascular cells have been proposed among possible explanations for the preferential location of AAA disease in the IR aorta, the mechanism(s) for this is still not known.

Objective:
To determine and compare the proteomes of the IR vs SR aorta regions in naïve mice and during KD vasculitis induced AAA development.

Methods and Results:
By using data-independent mass-spectrometry, we compared the proteomes of the IR and SR aortas isolated from control naïve mice and LCWE-injected KD vasculitis mice. Proteomic analysis revealed significant differential expression of 340 proteins between the IR aorta from control vs KD mice, and 407 differentially expressed proteins between the IR and SR aorta regions of KD mice. The differentially expressed proteins include those involved in cytoskeletal reorganization, cell motility and adhesion as well as acute phase and oxidative stress responses. Ingenuity Pathway Analysis reveals the activation of key regulators of innate immune responses as well as the ER Stress response. TGF-β1, IL-6, and IL-1β are regulators of several protein that were activated in the IR aortic tissues.

Conclusion:
Proteomic differences exist between normal AA vs AAA and in IR vs SR aorta regions isolated from mice developing AAA. We show that a broad inflammatory response is generated in the aneurysm which maybe critical for the development and progression of the AAA and may provide possible novel therapeutic targets. (Supported by AHA 17SDG33671141 to MNR and NIH R01AI07272607 to MA).
Abstract 274: Cd36-mediated Lipid Metabolism Promotes Endothelial Cell Angiogenic Function and Vascular Repair Ina Hindlimb Ischemia Mouse Model
Authors: Bou Khzam Lara , Son Ni-Huiping , Goldberg Ira J
Published: March 2019

Under physiological conditions, endothelial cells (ECs) play an important role in maintaining vascular integrity by acting as non-thrombogenic and non-inflammatory cells. ECs also mediate vascular repair and angiogenesis, a process by which new blood vessels are formed from pre- existing blood vessels. Hyperglycemia has been shown to alter EC angiogenic potential. However, few studies have investigated the effect of fatty acids (FAs) on EC growth and migration. CD36 is an important FA transporter expressed by ECs. We hypothesize that circulating FAs regulate angiogenic function in a CD36-dependent manner. To test this, we studied EC proliferation and migration of control and CD36 deficient mouse heart and lung ECs. siRNA- and ASO-mediated knockdown of CD36 in ECs treated with oleic acid (OA) did not affect EC proliferation. However, in wound healing experiments, OA significantly increased the migration of ECs, whereas CD36 knockdown prevented the OA-induced increase in wound healing potential. In EC transwell migration experiments, OA stimulation, which increased the recruitment and migration of ECs, was not found after CD36 knockdown. Thus, our
in vitro
results suggest that loss of CD36 hinders EC angiogenic function. In control mice, 21-day recovery post-hindlimb ischemia increased EC content in muscle, as assessed by measuring CD31 and MMP9 expression, compared to that found at baseline in normal muscle. Mice with EC-specific CD36 deletion also showed increased CD31 and MMP9 expression. However, no further increase was

found in EC-specific CD36 knockout ischemic muscle compared to normal muscle. Thus, our
in vivo
data suggest that the response to ischemia—increasing EC marker expression—is lost with EC-specific CD36 deficiency.
Abstract 277: Lung Gene Transfer With Sarcoplasmic Reticulum Calcium ATPase Prevent Disease Progression in Pulmonary Arterial Hypertension
Authors: Bueno-Beti Carlos , Katz Michael G M , Fargnoli Anthony S , Kohlbrenner Erik , Bridges Charles R , Sassi Yassine , Hajjar Roger J , Hadri Lahouaria
Published: March 2019

Pulmonary arterial hypertension (PAH) is a fatal disease characterized by vascular remodeling leading to high pulmonary arterial pressure (PAP) and right ventricular (RV) heart failure. Gene therapy is a promising approach to treat PAH. The most used monocrotaline (MCT) rat model of PAH does not mimic the pathophysiology of the PAH in humans. A refined rat model, the pneumonectomy plus MCT (PNT-MCT), displays all of the features of PAH and most importantly plexiform lesions whereby therapeutics aim to reverse. In this study, we investigated whether intratracheal delivery of sarcoplasmic reticulum calcium ATPase (SERCA2a) gene reverse the severity of PAH in the PNT- MCT model. Left pneumonectomy was performed in rats. One week later, the animals received the MCT injection. At 3 weeks, the severity of PAH disease was confirmed. Thereafter, PNT-MCT rats received either intratracheal delivery of gene construct with adeno-associated virus/SERCA2a (AAV1/SERCA2a) or saline. Hemodynamic parameters were determined by magnetic resonance imaging (MRI) and RV catheterization. RV hypertrophy, heart and lung fibrosis were assessed. Molecular biology assays for gene expression and immunostaining were used to quantify SERCA2a and disease markers. Four weeks after gene delivery, RV function was improved in AAV1.SERCA2a treated-animals with an increase of stroke volume and ejection fraction compared to saline group (275 ± 22 μl vs. 192 ± 22 μl and 56 ± 3 μl vs. 44 ± 3 μl, p<0.05), while RV end systolic volume was decreased (199 ± 16 μl vs. 283 ± 24 μl, p<0.05). Hemodynamic parameters including mean pulmonary pressure were improved in AAV1.SERCA2a group (26 ± 3 mmHg vs. 61 ± 6 mmHg and 21 ± 3 mmHg vs. 41 ± 3 mmHg respectively, p<0.01) compared to control. RV hypertrophy was reduced in AAV1.SERCA2a treated animals (0.37g ± 0.03 vs. 0.63g ± 0.02, p<0.0001). All serotypes of collagen demonstrated decreased expression after AAV1/SERCA2a administration. Histologically, the animals after gene therapy showed a significant regression of plexiform lesions from grade 4 to grade 1-2. In conclusion, intratracheal administration of AAV1/SERCA2a gene can reverse the severe PAH phenotype and may be considered as a potential treatment.