bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 397: The Interaction of Integrin beta1 to Galpha13 Mediates RhoA Inhibition and Cell Migration
Authors: Shen Bo , Estevez Brian , Kreutz Barry , Karginov Andrei , Mosher Deanne , Bai Yanyan , Qian Feng , Norifumi Urao , Du Xiaoping
Published: March 2022

Background:
Integrin-dependent cell migration is critically important in many physiological and pathological processes such as angiogenesis, inflammation, wound healing, and atherosclerosis. However, the underlying signaling mechanisms remain unclear.

Hypothesis:
We have recently shown that Ga13 directly interacts with the cytoplasmic domain of integrin b3 subunits in platelets and is a proximal mechanism of integrin outside-in signaling. We further showed that the binding site for Ga13 requires a conserved ExE motif also present in b1 integrins important in adhesion and migration of nearly all vascular cells. We hypothesize that the direct binding of Ga13 to the ExE motif in integrin b1 plays an important role in mediates b1-dependent cell migration.

Results:
To specifically study the role of b1 integrin in cell migration, we compared cell migration of GD25 cells that are deficient in integrin b1 subunit with cells that re-constitute b1 expression using a scratch wound healing assay and a transwell migration assay. Cell migration was abolished in b1-deficient cells, indicating a critical role for b1 in these assays. To study whether Ga13-integrin interaction is important in cell migration, we constructed b1 mutants that were deficient in binding to Ga13 by mutating the critically important glutamic acid residues to alanine. Co-immunoprecipitation experiments indicate the ExE to alanine mutants abolished Ga13-integrin interaction. The b1-dependent cell migration is dramatically inhibited in the ExE to alanine mutant b1-expressing cells. Importantly, a small peptide based on the ExE motif similarly inhibited cell migration. Furthermore, Ga13 binding-deficient b1 mutant cells showed diminished b1 integrin-dependent Src activation and accelerated RhoA activation during cell spreading on fibronectin, suggesting that the integrin-dependent transient inhibition of RhoA was abolished.

Conclusions:
Ga13 mediates integrin-dependent cell migration by direct binding to the ExE motif of integrin b1 and mediating c-Src activation and RhoA inhibition.
Abstract 416: G Protein-coupled Receptor 15 is Associated with Inflammation, Smoking and Ischemia
Authors: Haase Tina , Müller Christian , Schillert Arne , Lindner Diana , Schnabel Renate , Wild Philipp , Lackner Karl , Münzel Thomas , Westermann Dirk , Ziegler Andreas , Blankenberg Stefan , Zeller Tanja ,   German Cntr for Cardiovascular Rsch (DZHK)
Published: March 2022

Introduction:
The mechanisms leading to myocardial infarction (MI) are still not fully understood. As the orphan G protein-coupled receptor 15 (
GPR15
) gene has recently been shown to be hypomethylated in smokers as well as to be involved in inflammation, it poses a new candidate gene for cardiovascular disease.

Hypothesis:
We hypothesized that GPR15 influences the development of MI via smoking and inflammation.

Methods:
To investigate the effect of smoking and changes in expression over time, monocytic
GPR15
mRNA expression was measured in smokers (n = 273), ex-smokers (n = 533) and never-smokers (n = 687) from the Gutenberg Health Study (GHS) via qPCR.

In order to assess
GPR15
expression after MI, mRNA expression in peripheral blood mononuclear cells was compared between 112 young MI cases (< 50 years) and 112 controls from the GHS using the Affymetrix Exon ST1.0 GeneChip Array. Subsequently, cardiac
GPR15
expression in mouse models of MI (n = 8), myocarditis (n = 8), hypertrophy (n = 6) and untreated controls was measured via qPCR.

Results:
GPR15
expression was significantly increased in smokers compared to never-smokers (16.0 ± 2.4-fold, p < 0.001). Ex-smokers still had significantly elevated
GPR15
expression levels compared to never-smokers up to ten years after quitting (3.9 ± 0.9-fold, p < 0.001).

Furthermore,
GPR15
expression was 1.43-fold higher in young MI patients compared to controls (± 0.09-fold, p = 1.9*10
-7
). Likewise, mouse
Gpr15
expression was significantly elevated in infarcted hearts (6.3 ± 1.1 fold, p < 0.05) and in inflamed hearts (10.2 ± 2.5 fold, p < 0.01) but not in the hypertrophic hearts (1.0 ± 0.3) compared to controls.

Conclusion:
Consistent with literature on
GPR15
methylation,
GPR15
expression was increased in smokers, but also after inflammation and ischemia, independently of smoking. In conclusion, GPR15 might play a role in the development of myocardial infarction via smoking and inflammatory mechanisms.
Abstract 434: Formin Dependent Regulation of Adherens Junction Assembly
Authors: Mumal Iqra , Copeland John W
Published: March 2022

Adherens junctions are cadherin-dependent structures that mediate intercellular signaling and structural integrity. In endothelial cells, adherens junctions are the primary determiners of vascular permeability and endothelial barrier function. Adherens junctions connect directly to the actin cytoskeleton and this connection is essential for junction formation and maintenance. Formins are a highly conserved family of cytoskeletal remodeling proteins whose activity has been implicated in regulating junction formation in other cell-types. Therefore, we tested the hypothesis that formin activity is essential for adherens junction assembly in endothelial cells. A small-molecule formin inhibitor (smiFH2) was used to determine the effect of formin inhibition on junction formation in Telomerase-Immortalized Microvascular Endothelial (TIME) cells. Using an in vitro vascular permeability assay, we determined that smiFH2 treatment caused a dose-dependent inhibition of junction formation. We used siRNA to knockdown expression of seven of the formins expressed in TIME cells in order to determine which specific formins were required to maintain endothelial barrier function. We determined that individual knockdown of three formins, FHOD1, FHOD3 and Dia1, significantly increased the permeability of the endothelial monolayer. Interestingly, the knockdown of a fourth formin, FMNL2, had the opposite effect and actually potentiated barrier function. Knockdown of the remaining formins had little or no effect on junction formation. Knockdown of FHOD3 had the greatest inhibitory effect on junction assembly and VE-cadherin, β-catenin and α-catenin protein levels were decreased in FHOD3-depleted cells. The FHOD3 knockdown cells were also elongated in comparison to controls and formed thin linear adherens junctions and few focal adherens junctions. In contrast, the morphology of FMNL2-depleted cells did not appear obviously different from controls. In conclusion, our results suggest that multiple formins play diverse roles in endothelial cell adherens junction formation and maintenance.
Abstract 446: Ebp50 Regulates Oxidative Stress Through Skp2 and Foxo1
Authors: Procaccia Vera , Leslie Kristen L , Malik Erum Z , Bisello Alessandro
Published: March 2022

Inflammation and oxidative stress play important roles in cardiovascular disease. Reactive oxygen- and nitric oxide (NO)-derived species affect proliferation and migration of endothelial and vascular smooth muscle cells (VSMC), and amplify vascular inflammation. In this work we explore the role of Ezrin-Radixin-Moesin (ERM) binding protein of 50kDa (EBP50) as a novel mediator of oxidative stress. EBP50 plays a critical role in vascular remodeling. We reported that EBP50 mediates migration and proliferation of VSMC. In addition, EBP50 potentiates inflammatory responses (including iNOS expression) in macrophages and vascular cells. EBP50 stabilizes S-phase kinase associated protein 2 (Skp2) - a substrate recruiting component of the E3 ubiquitin ligase that targets FoxO1 for degradation. FoxO1 is a transcription factor that controls the expression of two key antioxidant enzymes - manganese superoxide dismutase (MnSOD) and catalase. Therefore, we hypothesize that EBP50 regulates oxidative stress through Skp2-FoxO1 axis. Our findings show a significantly greater nuclear localization of FoxO1 in EBP50 depleted VSMC compared to WT VSMC. Moreover, EBP50 knockdown increased FoxO1 stability following cycloheximide treatment. Consistent with these observations, expression of MnSOD and catalase were higher in EBP50 knockout primary macrophages and VSMC. To begin addressing the role of EBP50 on oxidative stress in vivo we used bone marrow transplants in LDLR-/- mice. Following reconstitution, mice were place on a high cholesterol diet for 12 weeks. We observed a significant 25% reduction in plaque area in EBP50-deficient marrow recipients compared to WT marrow recipients. In these mice there was a significant reduction in 4-hydroxynonanal (a marker of oxidative stress) and of 3-nitro-tyrosine (a marker of oxygen- and NO-derived reactive species) staining in the atherosclerotic plaques. Collectively, these observations suggest that EBP50, by regulating the localization and Skp2-mediated degradation of FoxO1, increases oxidative stress and inflammation during atherosclerosis. These studies further highlight the actions of EBP50 in controlling multiple signaling pathways that play a critical role in the pathogenesis of vascular disease.
Abstract 449: ApoA-I Suppresses Neointimal Hyperplasia Following Stent Deployment and Modulates Neointimal Cellular Phenotype
Authors: Vanags Laura Z , Tan Joanne T , Wise Steven G , Ali Ziad , Bursill Christina A
Published: March 2022

Background:
Increases in smooth muscle cell (SMC) cholesterol content reduce the expression of SMC phenotypic markers and increase macrophage related markers. The balance of neointimal SMCs to macrophage-like cells may influence neointimal hyperplasia following stent deployment. Apolipoprotein (apo) A-I, the main protein component of HDL, mediates the efflux of cholesterol from cells via the cholesterol transporter ABCA1. Through cholesterol loading of SMCs in vitro and in murine models of balloon angioplasty and stenting, we aimed to investigate the effect of apoA-I on SMC cellular phenotype and neointimal hyperplasia.

Methods and Results:
SMCs loaded with cholesterol (Cholesterol:MβCD complex) showed a reduction in SMC α-actin mRNA. Incubation with apoA-I restored SMC α-actin mRNA to control levels. Thoracic aortic segments from apoE-/- mice underwent balloon angioplasty or stenting then were carotid-interposition grafted into recipient mice. Mice received three PBS or apoA-I (40mg/kg) injections prior to surgery and then on alternate days until sacrifice (28 days). There were no changes in total cholesterol, LDL and HDL between groups. Thrombosis rates were 16% lower in apoA-I injected mice. Histological analysis revealed that stented aortic sections from apoA-I injected mice had smaller neointimal areas (18%±6), compared to control mice p<0.05. SMC α-actin staining was strikingly higher in the neointimas of apoA-I stented arteries (76%±34, p<0.05), while RT-PCR analysis of balloon angioplasty injured aortas found mRNA levels of macrophage marker CD68 were lower in apoA-I infused mice (30%±8, p<0.01). Furthermore, mRNA levels of ABCA1 and the cholesterol efflux regulator PPAR-γ were significantly higher in apoA-I aortic segments (30%±13 and 48%±20, respectively), p<0.05.

Conclusion:
ApoA-I infusions reduce neointimal area following stent deployment. Neointimas of apoA-I infused mice had higher SMC α-actin and lower macrophage CD68 expression. Mechanistically, this may be via increased SMC cholesterol efflux as apoA-I increased the expression of ABCA1 and PPAR-γ in balloon-injured arteries. These findings have significant implications for therapeutic modulation of neointimal hyperplasia following stent deployment.
Abstract 45: Characterization of a Novel Integrin Binding Protein that is Essential for αIIbβ3 Outside-in Signaling and Hemostasis
Authors: Xiang Binggang , Zhang Guoying , Ye Shaojing , Huang Cai , Liu Jun , Tao Min , Ruan Changgeng , Smyth Susan , Whiteheart Sidney , Li Zhenyu
Published: March 2022

Integrins are heterodimeric (α/β) membrane proteins that play fundamental roles in many biological processes,
e.g.
cell adhesion and spreading, which are important for platelet function and hemostasis. Integrin function is modulated by bi-directional transmembrane signaling: inside-out and outside-in, which is mediated through the interactions between integrin cytoplasmic tails and intracellular, regulatory proteins. Here, we show that VPS33B, a member of the Sec1/Munc18 (SM) family and component of the Class C core of the CORVET/HOPS sorting complexes, binds directly to the β subunit. Overexpression of VPS33B in CHO cells potentiated α
IIb
β
3
outside-in signaling but not inside-out signaling. Platelets, from megakaryocyte- and platelet-specific VPS33B conditional knockout mice we generated, had normal morphology yet their spreading on fibrinogen was impaired and they failed to support clot retraction. Platelet aggregation and ATP secretion in response to low-dose thrombin were reduced in the VPS33B knockout mice. α
IIb
β
3
-mediated endocytosis of fibrinogen was also defective. Tail bleeding times were prolonged in the VPS33B knockout mice. Furthermore, VPS33B acted upstream of the RhoA-ROCK-MLC and Rac1 dependent pathways that leads to clot retraction and cell spreading, respectively. Our work demonstrates that vesicular trafficking complexes are a novel class of modifiers of integrin function and in hemostasis. Our data also provide insights into the molecular mechanism and treatment of ARC syndrome.
Abstract 456: Hypercholesterolemia Induced by a PCSK9 Gain-of-function Mutation Augments AngII-induced AAAs in C57BL/6 Mice
Authors: Balakrishnan Anju , Howatt Deborah A , Cassis Lisa A , Daugherty Alan , Lu Hong
Published: March 2022

Objective:
Angiotensin II (AngII)-induced abdominal aortic aneurysms (AAAs) have increased incidence in hyper- versus normocholesterolemic mice. The purpose of this study was to determine whether infection of C57BL/6 mice with an adeno-associated viral vector (AAV) expressing a gain-of-function mutation of mouse proprotein convertase subtilisin/kexin type 9 (PCSK9) to induce hypercholesterolemia would augment AngII-induced AAAs.

Methods and Results:
Male C57BL/6 mice (8 weeks old; The Jackson Laboratory) were assigned randomly to groups infected with AAVs containing either an empty vector or a mouse PCSK9 gain-of-function (D374Y) mutation. Mice received a single intraperitoneal injection of AAV (n=10/group at a dosage of 10 x 10
10
genomic copies/mouse) and were fed a saturated fat-enriched diet for 6 weeks. Two weeks after AAV injection, mice were infused with AngII (1,000 ng/kg/min) for 28 days. Plasma PCSK9 concentrations were increased profoundly 2 weeks after injection of AAVs containing the PCSK9 mutation and remained elevated during the study (P<0.001). Expression of the PCSK9 mutant significantly increased plasma total cholesterol concentrations to greater than 800 mg/dl within 2 weeks that were maintained through 4 weeks of AngII infusion. Increased plasma cholesterol concentrations were due to increased VLDL and LDL cholesterol, as determined by size exclusion chromatography. In agreement with high plasma cholesterol concentrations, atherosclerotic lesion area of the aortic arch region was increased profoundly by the PCSK9 mutant (P<0.001). AAAs were quantified by ex vivo maximal aortic width of the suprarenal aorta. There was a significant increase of maximal aortic width of abdominal aortas (P=0.005) in AngII-infused mice expressing the PCSK9 mutant.

Conclusion:
AAV-mediated expression of a PCSK9 gain-of-function mutation is a rapid, easy, and efficient approach to induce hypercholesterolemia and promote AngII-induced AAAs in C57BL/6 mice.
Abstract 482: Signaling Lipid Lysophosphatidic Acid Is a Critical Link to Diet-induced Obesity, Cellular Bioenergetics and Breast Cancer Angiogenesis
Authors: Dong Liuyi , Yuan Ye , Aguilera-Barrantes Irene , Chen Yiliang , Sturich Adrian , Yuan Rong (Gloria) , Wu Shiyong , Silverstein Roy , Ren Bin
Published: March 2022

Background:
Obesity increases breast cancer (BC) risk but mechanisms by which obesity promotes BC progression remain largely unknown. Nutritional obesity is accompanied by increasing phospholipid lysophosphatidic acid (LPA), which stimulates angiogenesis by turning off CD36 antiangiogenic switch in microvascular endothelial cells (MVECs) via protein kinase D (PKD-1) signaling pathway that was reported to regulate mitochondrial function. CD36 impacts cellular energy metabolism as it is known to function as a fatty acid transporter. Hypothesis Obesity-derived LPA promotes angiogenesis and BC progression by modifying mitochondrial bioenergetics in cancer and endothelial cells via PKD-1 signaling.

Methods:
MVECs and breast adenocarcinoma cells were used for elucidating key signaling pathway and detecting mitochondrial respiration. BC angiogenesis model was established in diet-induced obese and lean mice.

Results:
LPA enhanced mitochondrial respiration in breast adenocarcinoma cells transduced with PKD-1, leading to elevations in oxygen consumption rate (OCR) and ATP-linked OCR, but not in extracellular acidification rate (ECAR). The cancer cells exposed to conditional medium from MVECs transduced with PKD-1 showed reduced level of basal OCR, ATP-linked OCR, and ECAR. Tumor-associated ECs exposed to LPA decreased CD36 expression whereas LPA exposure reduced not only basal OCR and ATP-linked mitochondrial respiration but also basal ECAR. Overexpressing PKD-1 also reduced basal OCR and ATP-linked mitochondrial respiration in these cells. In syngeneic breast adenocarcinoma model, BC grew larger in diet induced obese mice when compared with that in lean controls (p < 0.01). The tumor endothelium in diet-induced obese mice showed increased LPA receptor 1 expression, along with reduced CD36 expression and enhanced PKD-1 phosphorylation. Concurrently, the quantity of periphery vessels was increased, with elevations in plasma level of angiogenic proteins leptin and PAI 1.

Conclusion:
LPA/PKD-CD36 signaling may regulate angiogenesis and promote BC progression by controlling mitochondrial respiration via modulating cellular bioenergetics. Targeting LPA/PKD-CD36-metabolic signaling axis might provide a novel therapeutic strategy.
Abstract 503: Osteoporosis and Aterial Calcification: Are These Processes Related?
Authors: Miralles Manuel , Arrebola Manel , Plana Emma , Sanchez-Nevarez Ignacio
Published: March 2022

The relationship between arterial calcification and osteoporosis has been referred to in several studies. However, its temporal relationship has scarcely been analyzed.

Hypothesis:
We assessed the hypothesis that temporal evolution of Ca content in the carotid bifurcation is related with densitometric variation in bone calcium mass, in order to assess the possible connection of both processes through the Ca/P metabolism.

Patients and methods:
Design:
Prospective cohort study.
Population
: 32 asymptomatic patients with stenosis of the internal carotid artery (ICA)>50% (duplex scanningr).
Determinations:
Ca content (quantitative Angio-CT) in 3D reconstruction of the carotid bifurcation, basal and at 12+/-2 months. Bone densitometry of spine and femur, basal and 12+/-2 months, biochemistry and Ca-P metabolism (Ca, P, vit.D, PTH, OPG, OPG).
Statistical analysis:
Descriptive statistics, comparison of means (t-test), uni-and multivariate linear regression (Intraplaque Ca/ bone densitometry, Ca-P metabolism).

Results:
An increase of intraplaque Ca respect to baseline values (79.2 (55) vs 64.58 (49.2) mm
3
.xU.H., p = 0.12) and a significant decrease in bone mass respect to baseline determination (2.08 (0.22) vs 2.06 (0.23) g/cm
2
, p = 0.013) were observed. However, a positive correlation was identified between the relative variation of the Ca content and bone mass (r= 0.574, p= 0.003). In other words, a greater tendency to osteoporosis was associated with a reduced tendency to intraplaque calcium progression. In Addition, an inverse relationship between plasma levels of Vit D and basal densitometry (r= -0.459; p=0.036) were found. Only P concentration in urine showed a moderate positive correlation with increased intraplaque Ca (r = 0.488, p = 0.015). No other variable, was related to arterial calcification.

Conclusions:
As in other published series, an independent tendency to progressive arterialcalcification and bone decalcification was found in this study. However, these results suggest a direct temporal relation in calcium deposit evolution of both localizations, so it is not possible to rule out that their modulation is governed by similar activation and regulation mechanisms.
Abstract 532: The Receptor for Advanced Glycation End Products (RAGE) Suppresses Macrophage Cholesterol Transport in Diabetes
Authors: Daffu Gurdip , Shen Xiaoping , Abedini Andisheh , Senatus Laura , Hurtado del Pozo Carmen , Rosario Rosa , Song Fei , Friedman Richard , Ramasamy Ravichandran , Schmidt Ann Marie
Published: March 2022

Diabetes is a leading cause of cardiovascular morbidity and mortality. In human subjects, diabetes suppresses macrophage cholesterol efflux capacity, at least in part via reduction in levels of the major cholesterol transporters, ABCA1 and ABCG1. We tested the hypothesis that the receptor for advanced glycation end products (RAGE) contributes to these processes. We report that ligand-RAGE interaction suppresses cholesterol efflux to ApoA1 and HDL in primary murine bone marrow-derived macrophages (BMDMs) and in human THP-1 cells, at least in part via downregulation of ABCA1 and ABCG1. In vivo, compared to cholesterol-loaded wild-type (WT) diabetic murine BMDMs, diabetic BMDMs devoid of Ager (gene encoding RAGE) displayed significantly higher reverse cholesterol transport (RCT) to plasma, liver and feces when injected into WT non-diabetic mice. In atherosclerotic plaques from mice devoid of both Ldlr and Ager and fed a high cholesterol diet, higher Abca1 and Abcg1 mRNA transcript levels were also observed compared to Ager-expressing LDL receptor null mice. RAGE ligand AGEs suppress ABCG1 promoter luciferase activity and transcription of ABCG1 through reduced binding of PPARgamma (PPARG) to PPARG-responsive elements in the ABCG1 promoter. These data reveal that RAGE contributes to dysregulation of macrophage cholesterol metabolism, particularly in diabetes. We infer that antagonism of RAGE may mitigate accelerated atherosclerosis in the diabetic state, at least in part due to modulation of impaired cholesterol transport.
Abstract 582: Overexpression of Mnsod Reduces Aortic Valve Calcification Through Repression of Pro-osteogenic Signaling
Authors: Roos Carolyn M , Hagler Michael A , Versoza Grace C , Z Bin , Thalji Nassir M , Richardson Arlan G , Miller Jordan D
Published: March 2022

Increased BMP signaling and markers of osteogenic differentiation are strongly associated with regions of valvular calcification in aortic valves from humans with end-stage calcific aortic valve stenosis. While oxidative stress is also markedly elevated in these regions, it is unknown whether increasing antioxidant capacity can reduce pro-osteogenic signaling and valvular calcification. Therefore, we hypothesized that overexpression of manganese superoxide dismutase (MnSOD) can reduce pro-osteogenic signaling in the aortic valves from hypercholesterolemic mice, ultimately resulting in reduced valvular calcification and improved valvular function. We used Ldlr
-/-
/ApoB
100/100
(LA) mice that were wild-type for MnSOD (LA-MnSOD
0/0
) or overexpressing MnSOD (LA-MnSOD
Tg/0
) and fed a western diet (TD88137) for six months. We first measured mRNA levels of the BMP ligands BMP2 and BMP4 (qRT-PCR), both of which were unchanged between LA-MnSOD
0/0
and LA-MNSOD
Tg/0
mice. While there was a small but significant increase in Runx2 mRNA levels in LA-MnSOD
Tg/0
mice compared to wild-type littermates (1.22±0.09, 1.0±0.09, qRT-PCR, p < 0.05.) gene expression levels of late stage osteoblast marker Sp7 were reduced by approximately 50% in LA-MnSOD
Tg/0
compared to wild-type littermates (0.53±0.15, 1.0±0.24, qRT-PCR, p < 0.05). Sp7 protein levels (immunohistochemistry) were also significantly reduced in LA-MnSOD
Tg/0
compared to wild-type littermates (164.5±12.3 RFU vs 259.6±10.4 RFU, p < 0.05). Critically, aortic valve calcification (Alizarin Red) was also significantly reduced in LA-MnSOD
Tg/0
mice compared to their LA-MnSOD
0/0
littermates. Functionally, aortic valve cusp separation distance (high resolution echocardiography) was slightly improved in LA-MnSOD
Tg/0
mice compared to LA-MnSOD
0/0
mice (0.98±0.04 mm vs 0.91±0.03 mm, p = n.s.). Collectively, these data suggest that increasing mitochondrial antioxidant capacity can reduce pro-osteogenic signaling and attenuate aortic valve calcification, and may be a potential therapeutic target to slow the progression of valvular calcification and dysfunction in patients with calcific aortic valve stenosis.
Abstract 613: Fibroblast Growth Factor 2 Modulates Endotheliogenesis of Human Adipose Tissue Derived Stem Cells
Authors: Khan Sophia , Villalobos Miguel , Choron Rachel , Chang Shaohua , Brown Spencer , Carpenter Jeffrey , Liu Yuan , Tulenko Thomas , Zhang Ping
Published: March 2022

Background:
Adipose-derived stem cells (ASCs) are a potential adult mesenchymal stem cell source for restoring endothelial function in critical limb ischemia patients. Fibroblast growth factors (FGF) play a major role in angiogenesis and wound healing. The purpose of this study was to evaluate the effects of FGF and VEGF on human ASCs proliferation, migration and potential endothelial differentiation regards to their use as endothelial cell (EC) substitutes.

Methods:
ASCs were isolated from clinical lipoaspirates and cultured in M199 medium with FBS (10%), FGF2 (10ng/ml), VEGF (50ng/ml) or combinations of FGF2 and VEGF. Cell proliferation rates, viability, and migration were measured by growth curves, MTT and scratch assays. For cell attachment determinations, ASCs were seeded onto scaffold (small intestinal submucosa) for 5d. Endothelial differentiation capabilities of ASC were confirmed by expression of EC specific markers using qPCR, immunofluorescence staining and cord formation on Matrigel. PD173074, a selective inhibitor of FGF receptor, was used to confirm the importance of FGF signaling.

Results:
Increased proliferation rates were observed in ASCs treated with FGF2 when compared to ASCs cultured with VEGF and control groups (3.2 fold, p<0.05). In comparison with controls, FGF2 showed increased ASC migration (relative scratch width, p<0.01). FGF2-treated ASCs seeded onto the SIS scaffold for 5d exhibited increased attachment rates compared to ASCs cultured with VEGF (1.7 fold, p<0.01). After 10 days exposed to FGF, ASCs expressed EC-markers (CD31, vWF mRNA) and formed cords upon Matrigel. Cultured ASCs with FGF2 and VEGF increased eNOS mRNA and protein levels when compared to ASCs cultured with FGF2 or VEGF alone. Furthermore, blockade of FGF signaling with PD173074 blocked ASC EC differentiation potential.

Conclusions:
Our results indicate that: 1) FGF is an ASC promoter for proliferation, migration, attachment and endothelial differentiation; 2) FGF and VEGF have a co-stimulatory effect on ASC endotheliogenesis; and 3) FGF signaling mediates ASC EC differentiation. These results suggest that ASCs with enhanced FGF2 signaling may potentially be used for tissue engineering and cell-based therapies in critical limb ischemia patients.
Abstract 620: Water as a Universal Biosensor for Inflammation, Insulin Resistance and Dyslipidemia
Authors: Robinson Michelle D , Deodhar Sneha , Mishra Ina , Patel Vipulkumar , Gordon Katrina , Vintimilla Raul , Johnson Leigh , O’Bryant Sid , Cistola David P
Published: March 2022

Current proteomic strategies exploit the measurement of hundreds-to-thousands of potential biomarkers to establish profiles of disease risk. We propose an alternative strategy - inverse proteomics - which utilizes just one marker to monitor the status of many biomolecules all at once. Water is an attractive surveillance system, as it forms hydrogen bonds with virtually every protein, lipoprotein and metabolite in human blood. Here we show that the mobility of water in plasma and serum samples from apparently healthy human subjects correlates with known markers of inflammation, insulin resistance, dyslipidemia and possibly, oxidative stress. Water mobility is assessed in a six-minute experiment that requires no sample manipulations or chemical reactions. Rather, the spin-spin relaxation time constant (T
2
) is measured non-invasively using benchtop time-domain nuclear magnetic resonance. The current discovery provides a framework for developing a simple blood test that could identify individuals with hidden risk for diabetes, atherosclerosis and Alzheimer’s disease.
Abstract 632: Premature Aortic and Carotid Stiffness in Systemic Lupus Erythematosus: Which One is First or Worse?
Authors: Roldan Paola , Greene Ernest , Roldan Pablo , Rodriguez Rodrigo , Roldan Carlos
Published: March 2022

Background:
Increased aortic and carotid stiffness are common in patients with systemic lupus erythematosus (SLE) and are associated with increased morbidity and mortality. Although the aorta and carotid arteries have different anatomy, hemodynamics, and cardiovascular disease implications, it is unknown whether stiffness affects these arterial beds in a parallel or divergent fashion.

Methods:
43 SLE patients (41 women, age 36 ± 11 years) and 17 age-and-sex matched healthy volunteers (14 women, age 34 ± 12 years) underwent clinical and laboratory evaluations and multiplane transesophageal echocardiography and carotid ultrasonography for assessment of aortic and carotid stiffness, respectively. Stiffness at the proximal, mid, and distal thoracic aorta and stiffness of the right common carotid artery were assessed using a standard Pressure-Strain Elastic Modulus, defined as = k (sBP – dBP)/[(sD - dD)/dD)] where k = conversion factor from mmHg to Nm
-2
(Pascal units), sBP = systolic blood pressure, dBP = diastolic blood pressure, sD = systolic diameter, and dD = diastolic diameter.

Results:
Aortic stiffness was significantly higher in SLE patients than in controls after adjusting for age and atherogenic risk factors (7.96 ± 4.16 versus 5.77 ± 2.32 units, p = 0.01). However, carotid stiffness was similar in patients and controls (6.39 ± 2.70 versus 5.55 ± 2.68 units, p = 0.29). In SLE patients, aortic stiffness was significantly higher than carotid stiffness (7.96 ± 4.17 versus 6.39 ± 2.70 units, p = 0.02). In relation to age at diagnosis of SLE, aortic stiffness occurred earlier and was greater than carotid stiffness (RM ANCOVA, p = 0.03).

Conclusion:
Aortic stiffness is higher and precedes carotid stiffness in young adult SLE patients. These findings of an accelerated and divergent pathogenesis of aortic as compared to carotid stiffness may be explained by SLE-related chronic or recurrent autoimmune-mediated inflammation and thrombogenesis superimposed on the severe anatomic curvature, higher pulse pressure, and higher reciprocating shear stresses of the aorta as compared to the nearly straight and less oscillating shear forces of the carotid arteries.
Abstract 634: Hypercholesterolemia Increases the Incidence of Colorectal Neoplasia by Impairing Hematopoietic Stem Cell Differentiation Towards Nkt and γδ T Cells
Authors: Tie Guodong , Yan Jinglian , Messina Julie , Messina Louis
Published: March 2022

Rationale:
Hypercholesterolemia, a co-morbidity of obesity, is one of the most common diet-related metabolic disorders and is an independent risk factor for colorectal neoplasia. As yet, no generalizable mechanism has been described to show how hypercholesterolemia increases the risk of cancer especially that of colorectal neoplasia.

Objective:
We hypothesized that hypercholesterolemia impaired the differentiation of hematopoietic stem cells (HSCs) toward the cellular components critical to immunosurveillance against colorectal neoplasia. Identifying the mechanisms by which hypercholesterolemia affects HSC differentiation may provide clues to understanding how hypercholesterolemia increases all-cause mortality and especially that of colorectal cancer in divergent clinical studies.

Methods and Results:
Two different hypercholesterolemic mice were applied in the study, including ApoE-/- mouse and high cholesterol diet fed WT mouse. Colorectal neoplasia was induced by IP injection of AOM. The incidence of colorectal neoplasia was significantly higher in these hypercholesterolemic mice. FACS analysis showed that NKT and γδT cells were significantly fewer in the thymus and colon of hypercholesterolemic mice. The infiltration of NKT and γδT cells in tumors isolated from hypercholesterolemic mice was attenuated. In agreement, in vitro differentiation assay demonstrated that the differentiation capacity of HSCs towards NKT and γδT cells was significantly impaired in the groups isolated from hypercholesterolemic mice. The deficiency in NKT or γδT cells increased the incidence of AOM-induced colorectal neoplasia.

Conclusion:
NKT and γδT cells are critical cellular components in the immnuosurveillance against colorectal neoplasia. Hypercholesterolemia increases the incidence of colorectal neoplasia by impairing the differentiation of HSCs towards NKT and γδT cells.
Abstract 661: Tie2 Deficiency Leads to Lymphatic Defects and Resistance to High-fat Diet Induced Obesity
Authors: Yamaguchi Shinji , Thomson Benjamin R , Heinen Stefan , Economides Aris N , Quaggin Susan E
Published: March 2022

The Angiopoietin-Tie2 signaling pathway is a critical regulator in vascular development, with conventional Ang1 or Tie2 knockout mice dying in utero with vascular defects. While the role of this pathway in vascular development is apparent, the severity of developmental phenotypes makes it difficult to study Tie2 in vivo. Here, we investigate the functional role of Tie2 in embryonic as well as adult mice using inducible knockout mouse models (Tie2 cKO).

Tie2 inactivation at early stages of gestation (E12.5 or E14.5), which coincides with the initial stages of lymphatic development and remodeling, resulted in severe subcutaneous edema in E16.5 and altered lymphatic vessel morphology in the dermis of E17.5. These results indicate the important role of Tie2 in lymphatic development. In contrast, Tie2 inactivation at later stages (E18.5-P0) resulted in the birth of viable pups, indicating that Tie2 is dispensable after embryonic development. Adult male Tie2 cKO mice have similar blood glucose, glucose tolerance, and plasma lipids compared to controls. However, body weight of Tie2 cKO mice upon high-fat diet feeding was lower than that of control mice (47.5+-0.6g (control) vs. 38.0+-2.0g (Tie2 cKO), p<0.01 (4weeks after high-fat diet feeding)). Although plasma lipids (cholesterol and triglyceride) in control and Tie2 cKO mice were similar after high-fat diet feeding, mRNA expression of TNFa (100+-14% (control) vs. 40+-1.4% (Tie2 cKO), p<0.05) and F4/80 (100+-13% (control) vs. 53+-7.3% (Tie2 cKO), p<0.05) in epididymal fat were reduced in Tie2 cKO mice.

Our data show that Tie2 plays an import role in lymphatic development during embryogenesis and that Tie2 signaling is also important for adipose tissue growth in adult mice.
Abstract 694: The Impact of Glucose-Lowering Therapy and Lifestyle-Related Factors on Vascular Disease Risk Reduction in Younger and Older Adults: National Trend and New Evidence from Big Data
Authors: Liu Longjian
Published: March 2022

Objective:
To examine the independent and interaction impacts of glucose-lowering therapy and lifestyle-related factors (obesity and hypercholesterolemia) on vascular disease risk reduction in patients with type 2 diabetes mellitus (T2DM).

Methods:
Data from 1999 to 2012 National Health and Nutrition Examination Surveys in participants aged≥30 years old (n=29064) were analyzed. T2DM was classified by self-reported physician diagnosis and those who were under glucose-lowering therapy. Coronary heart disease (CHD), stroke, and hypercholesterolemia (H-TC) were classified by self-reported physician diagnosis. Obesity was defined by BMI≥30 (weight(kg)/height squared(m
2
)).

Results:
The prevalence of T2DM significantly increased from 8% in 1999 to 12.3% in 2012 in subjects aged <65 (p<.001), and from 19.8% to 26.1% in subjects aged≥65 (p<.0001). In logistic regression analysis, after adjustment for age, sex, race, education, obesity and H-TC, T2DM patients with glucose-lowering therapy had significantly lower risk of CHD (OR=0.76, 95%CI: 0.60-0.97, p=0.025) than those without glucose-lowering therapy. A further stratification analysis indicated a significant combined and dose-response effect of glucose-lowering therapy and body weight on the risk of CHD. Obese patients without glucose-lowering therapy had the highest prevalence rate of CHD, and non-obese patients with glucose-lowering therapy had the lowest rate in patients aged<65 (17.7% vs. 10.9%, a 38% reduction, p=0.035), and in those aged≥65 (37.0% vs. 28.0%, a 24% reduction, p=0.029). A significant effect of glucose-lowering therapy with non H-TC on CHD was also observed. A similar protective effect of glucose-lowering therapy with non-obesity and non H-TC on stroke was observed as well, although this association did not reach statistically significant.

Conclusion:
using a 14-year nationally representative data, the study provides new evidence that a drug-lifestyle interaction effect of glucose-lowering therapy and diet-related factors plays a significant role in CHD risk reduction in patients with T2DM, and this effect could be even greater in younger adults than that in the older.
Abstract 711: In Hospital Complications After PCI After Insertion of DES versus BMS in the United States
Authors: Khoshchehreh Mahdi
Published: March 2022

Background:
We evaluated the potential differences in patient complications and in-hospital mortality for patients who underwent primary percutaneous coronary intervention (PCI) with insertion of drug-eluting coronary artery stent (DES) versus those with insertion of bare metal stent (BMS) in the United States.

Method:
We used the Nationwide Inpatient Sample to examine differences in all-cause in-hospital mortality between DES versus BMS patients with a principal diagnosis of STEMI, NSTEMI and Coronary atherosclerosis ICD-9 codes. Bivariate and adjusted logistic regression analysis were used to identify any associations with DES versus BMS with increased subsequent complications and in-hospital mortality.

Results:
We identified 9140 admissions with PCI as a primary intervention, of which 85.03% native coronary artery stenosis, 5.9% presented with NSTEMI, 4.9% had STEMI, and 4.0% revascularized due to graft stenosis. Patients with graft stenosis and STEMI had the highest rates of BMS insertion (62% and 49% respectively).

The odds of in-hospital mortality were higher for BMS insertion among all for different group. However, the association lost the statistical significance after adjusting for patient characteristics, except for patients with native coronary artery stenosis (OR =1.96 95% CI: 1.02-3.8).

Major complications, including vascular, neurological, and mechanical complications were higher for BMS insertion for NSTEMI and graft coronary atherosclerosis patients. However, BMS insertion associated with less complication in STEMI patients and native coronary atherosclerosis. the later didn’t reach the statistical significance level. Interestingly, there was no in-hospital mortality in our sample among male with DES insertion.

Conclusion:
The clinical context of PCI and choice of DES versus BMS, with BMS a preferential choice in patients with evidence of thrombus (STEMI) is reflected in downstream complications and in-hospital mortality.
Abstract 724: Effect of Arterial Calcification on Lower Extremity Ischemia Score is Independent of Occlusive Disease
Authors: Zettervall Sara L , Fleser Paul , Deas Dale , Marshall Andrew , Guzman Raul J
Published: March 2022

Objective:
Calcification in lower extremity arteries is associated with a higher risk of amputation and may reflect the amount of atherosclerotic plaque in lower extremity vessels. The association between calcification, occlusive disease, and severity of ischemia, however, has not been explored. We tested the hypothesis that the association between calcification and worsening limb status was mediated by concomitant occlusive disease.

Methods:
Rutherford ischemia categories, tibial artery calcification (TAC) scores, and the extent of occlusive disease were measured in 276 patients with or without symptomatic peripheral artery disease. Other factors included age, sex, race, and history of diabetes, tobacco use, hypertension, or hyperlipidemia. Patients with severe renal insufficiency (eGFR <30) and patients with aortoiliac disease or acute ischemia were excluded. Multivariable ordinal logistic regression models were constructed using Rutherford ischemia categories (0 - 6) as the dependent variable.

Results:
When both asymptomatic and symptomatic patients were included in our model, tobacco use, hypertension, occlusion index, and TAC scores were independently associated with increasing Rutherford categories. In multivariate analyses involving only those patients with symptomatic PAD, only the tibial calcification score (OR 2.06 (1.39-3.06), p<0.01) and tobacco use (OR 2.69 (1.03-6.99), p=0.04) maintained an independent association with worsening ischemia categories.

Conclusions:
Calcification is associated with worsening lower extremity ischemia categories in a way that is independent of the extent of arterial occlusive disease. Further research aimed at understanding the contribution of arterial calcification to ischemia in PAD is warranted, as are efforts to develop treatments to control it.
Periodontal Pathogens, Serum Lipids, and Lipid Peroxidation in a Population-Based Sample
Authors: Wu Tiejian , Trevisan Maurizio , Falkner Karen L , Grossi Sara G , Ho Alex , Genco Robert J
Published: March 2022

P33

Epidemiological studies have demonstrated a significant link between periodontal disease (PD) and increased risk of cardiovascular disease (CVD). This study was conducted to evaluate if lipid metabolism and peroxidation may represent potential pathways linking PD to CVD. The study sample included 747 randomly selected residents of Erie and Niagara counties of New York who had 6 or more teeth and no history of heart attack. Periodontal assessments included pocket depth (POD) and attachment loss (ATL). Periodontitis was defined by 2+ teeth with ATL≥6mm and 1+ teeth with POD≥5mm. Subgingival plaque was sampled and tested using immunofluorescence microscopic technique for the presence of four putative periodontal pathogens: Bacteriodes forsythus (BF), Porphyromouas gingivilis (PG), Prevotella intermedia (PI), and Campyglobacter recta (CR). Fasting blood sample was obtained and determinations of total and LDL cholesterol, triglycerides, and plasma thiobarbituric acid reacting substance (TBARS) were performed. In the sample as a whole, prevalence of specific bacteria in the subgingival plaque were 29.6% for BF, 8.3% for PG, 31.6% for PI, and 4.4% for CR. As expected, prevalence was much higher in participants with periodontitis. Presence of BF and CR (but not PI and PG) was associated significantly with higher levels of LDL cholesterol (Mean LDL cholesterol (mg/dl), 151.3 for BF + and 142.8 for BF -, p<0.05; and 158.4 for CR + and 144.7 for CR -, p<0.05). Presence of PG and CR (but not BF and PI) was significantly associated with higher TBARS (Mean TBARS (nmol/ml), 1.51 for PG + and 1.38 for PG -, p<0.05; and 1.57 for CR + and 1.38 for CR -, p<0.05). These associations remained statistically significant after adjustment by sex, age, education, body mass index, diabetes status, smoking and alcohol use. No significant association with bacteria was found for total cholesterol and triglycerides. In conclusion, subgingival presence of specific periodontal bacteria (bf, pg, and cr) was associated with higher serum LDL cholesterol and/or plasma TBARS. This suggests that lipid alteration and peroxidation may represent potential pathways in the link between PD and CVD.
Periodontal Infection and Risk of Myocardial Infarction: A Population-Based Case-control Study
Authors: Trevisan Maurizio , Grossi Sara G , Falkner Karen L , Wu Tiejian , Ho Alex , Baker Julie , Genco Robert J
Published: March 2022

P34

Epidemiological studies have demonstrated a significant association between periodontal disease and increased risk of cardiovascular disease. Periodontal organisms were hypothesized to be the underlying factor for the association. This study investigated the relation of myocardial infarction (MI) with both periodontal health status and presence of periodontal pathogens in subgingival plaque. The study sample included 171 MI cases and 574 controls who had at least 6 teeth and were residents of Erie and Niagara counties of New York. Periodontal assessments included pocket depth and attachment loss. Subgingival plaque was sampled and tested using immunofluorescence microscopic technique for the presence of Bacteriodes forsythus (BF), Porphyromouas gingivilis (PG), Prevotella intermedia (PI), and Campyglobacter recta (CR). Results: Mean (SD) pocket depth (mm) was 2.2 (0.7) for MI cases and 2.0 (0.5) for controls (p<0.01). Mean (SD) attachment loss (mm) was 2.6 (1.2) and 2.2 (0.7) for cases and controls (p<0.01), respectively. Presence of periodontal pathogens was significantly more frequent among MI cases than controls. For the four bacteria (BF, PG, PI, and CR), the prevalence (%) of subgingival bacterum presence was 45.0, 14.6, 39.8, and 12.3 among MI cases, and 26.8, 8.2, 31.2, and 5.1 among controls. After adjustment for sex, age, education, smoking status, alcohol use, body mass index, diabetes status, and dental plaque index using logistic regression, the odds ratios (95% confidence intervals) of MI were 1.62 (1.17-2.25) for pocket depth (per 1 mm change), 1.36 (1.10-1.69) for attachment loss (per 1 mm change), 1.93 (1.30-2.88) for BF, 1.41 (0.81-2.48) for PG, 1.18 (0.80-1.73) for PI, and 1.83 (0.96-3.52) for CR presence in subgingival plaque. In conclusion, this case-control study demonstrated that poorer periodontal status and presence of specific bacteria such as BF in subgingival plaque were associated with an increased risk of MI.
Cigarette Smoking Increases the Magnitude of Intracoronary Thrombus Observed During Acute Myocardial Infarction
Authors: Giri Satyendra , Mitchel Joseph , Kiernan Francis J , Hirst Jeffrey A , Clive Jonathan , Fram Daniel B , Simon Daniel , Popma Jeffrey J , Mittleman Murray A
Published: March 2022

P60

BACKGROUND: Smoking is a well known modifiable chronic risk factor for coronary disease. However, whether it can trigger acute myocardial infarction (MI) is unknown. To determine whether smoking predicts intracoronary thrombus burden in the infarct artery, we analyzed angiograms of 902 patients undergoing emergent coronary angioplasty for acute MI. METHODS: Patients underwent a structured standardized interview. Smokers (n= 348; 39%) were asked the time of last cigarette smoked prior to symptom onset. Quantitative indices of reference vessel diameter, segmental and cross-sectional intracoronary thrombus area, and ordinal TIMI thrombus grades were analyzed by 2 angiographers using a validated, computer-assisted program. RESULTS: Compared to nonsmokers, smokers were younger (54 ± 12 vs 65 ± 13 years; p < 0.001), more likely to be male (75% vs 65%; p = 0.003), normotensive (68% vs 44%;p < 0.001), physically active (36% vs 29%; p = 0.03), tended to present early (277 ± 91 vs 293 ± 98 minute; p = 0.02), have more single-vessel disease (37 vs 25%;p < 0.001), but with larger thrombus area (16.9 ± 17 vs 13.7 ± 17 mm

2

; p = 0.009). Intracoronary thrombus burden was larger when symptom onset was closer to the time of last cigarette, however, smokers had a greater chance of achieving complete reperfusion (TIMI grade 3 flow: 96% vs 90%; p=0.01). CONCLUSIONS: Despite less coexistent risk factors among smokers, smoking appears to increase thrombogenicity and is temporally related to the magnitude of intracoronary thrombus in the infarct-related artery. Thus, in addition to chronic effects, smoking may acutely trigger acute MI by enhancing thrombogenecity.

Table 1.