bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 199: Adipose-Derived Stem Cells from Diabetic Patients Undergo Endothelial Differentiation for Use in the Creation of a Tissue-Engineered Vascular Graft
Authors: Policha Aleksandra , Zhang Ping , Shue Bing , Smolock Amanda , Chang Lily , Jimbo Masaya , Tulenko Thomas , Moudgill Neil , Whisenhunt Anumeha , Rao Atul , Eisenberg Joshua , DiMuzio Paul
Published: March 2022

INTRODUCTION:
Surgical bypass and dialysis access creation are common procedures performed in diabetic patients. Only 55% of patients have autologous vascular tissue available for use as a conduit in these operations. We have developed an alternative conduit composed of a vascular scaffold seeded with autologous adipose-derived stem cells (ASC) differentiated into endothelial cells (EC) to create a non-thrombogenic lumen.

OBJECTIVE:
To examine ASC from diabetic (DM) versus non-diabetic (NDM) patients in terms of isolation efficiency, proliferation, commitment towards EC lineage, and seeding onto a vascular scaffold.

METHODS:
ASC were isolated from peri-umbilical liposuction specimens by collagenase dispersion (DM n=53; NDM n=145). Isolation efficiency was defined by number of ASC isolated per gram of adipose tissue. Proliferation was assessed by constructing growth curves over 14d. ASC were differentiated in endothelial growth medium for 3wk. Differentiation was determined by measuring EC-specific gene expression (CD31, vWF) using qPCR. Lastly, ASC were seeded onto decellularized vein, flow conditioned from 0-9 dynes over 5d, and examined using confocal microscopy.

RESULTS:
ASC isolation efficiency did not differ between DM and NDM patients (224,028 cells/gm vs. 259,345 cells/gm, respectively; P=0.21). The growth curves for DM (n=6) and NDM (n=6) ASC also appeared similar, with no significant differences observed in cells counts between days 1-14. After 1, 2, and 3wk in culture, no significant difference in CD31 or vWF expression between DM (n=6) and NDM (n=6) cells was observed. Finally, retention of ASC to vascular scaffolds under physiological shear stress was similar between the two groups.

CONCLUSIONS:
ASC are isolated from DM in quantities similar to those observed in NDM. These cells grow at rates similar to NDM ASC, exhibit differentiation into an EC phenotype, and line the lumen of a tissue engineered vascular graft. These results suggest that adipose tissue may prove to be a more reliable source of autologous stem cells than blood or bone marrow, where the presence of diabetes and other co-morbidities significantly decrease the availability of stem cells for use in regenerative therapies.
Abstract 2: Circadian Regulation of Intestinal Lipid Absorption by ApoAIV Involves Forkhead Transcription Factors A2/O1 and MTP
Authors: Pan Xiaoyue , Munshi Mohamed K , Iqbal Jahangir , Queiroz Joyce , Sirwi Alaa A , Shah Shrenik , Younus Abdullah , Hussain M Mahmood
Published: March 2022

Clock, MTP and nocturnin are involved in the circadian regulation of intestinal lipid absorption. Here, we clarified the role of apolipoprotein AIV (apoAIV) in the diurnal regulation of plasma lipids and intestinal lipid absorption in mice. Total plasma triglyceride/cholesterol in ApoAIV
-/-
mice showed diurnal variations similar to ApoAIV
+/+
mice; however, increases in plasma triglyceride at night were significantly lower in these mice. ApoAIV
-/-
mice absorbed fewer lipids at night and showed blunted response to daytime feeding. To explain reasons for these lower responses, we measured MTP expression; intestinal MTP was low at night and its induction after food entrainment was less in ApoAIV
-/-
mice. Conversely, apoAIV overexpression increased MTP mRNA in hepatoma cells indicating transcriptional regulation. Mechanistic studies revealed that sequences between -204/-775 bp in the MTP promoter respond to apoAIV and that apoAIV enhances expression of FoxA2/FoxO1 transcription factors and their binding to the identified
cis
-elements in the MTP promoter at night. Knockdown of FoxA2/FoxO1 abolished apoAIV mediated MTP induction. Moreover, FoxA2/FoxO1 expression showed diurnal variations and their expressions were significantly lower in ApoAIV
-/-
mice. These data indicate that apoAIV modulates diurnal regulation of lipid absorption by regulating forkhead transcription factors and MTP, and that inhibition of apoAIV expression might reduce plasma lipids.
Abstract 200: Inhibition of Four-and-a-Half Lim Domain Protein 2 Increases Survival and Migratory Capacity of Human Early Outgrowth Endothelial Progenitor Cells Through Upregulation of Sphingosine Kinase-1: Implications for Endothelial Regeneration
Authors: Ebrahimian Talin , Arfa Omar , Simeone Stefania , Lehoux Stephanie , Wassmann Sven
Published: March 2022

Background:
Inhibition of Four-and-a-half LIM domain protein-2 (FHL-2) attenuates atherosclerotic lesion formation and increases endothelial cell migration. Endothelial progenitor cells (EPCs) substantially contribute to endothelial repair. We investigated the role of FHL-2 in the regulation of early outgrowth EPC number and function.

Methods and Results:
Early outgrowth EPCs were obtained from human peripheral blood. FHL-2 knockdown in EPCs by small-interfering RNA (siRNA) resulted in a significant increase in EPC number and a reduction of apoptosis (by 40%), as indicated by a decrease of cleaved caspase-3, through activation and translocation to the membrane, of sphingosine kinase-1 (SK-1), enzyme that metabolizes sphingosine- 1 phosphate (s1p). Furthermore, FHL-2 siRNA increased significantly (2 fold) stromal derived factor (SDF) -1- induced EPC migration; through upregulation of α-v/β-3 and α-v/β-5 integrins; this was associated with an increase of the F-actin binding protein cortactin, known to promote migration. Interestingly, increased SDF-1- induced EPC migration and upregulation of cortactin by FHL-2 siRNA were totally prevented by
CAY10621
, a specific inhibitor of SK-1. In addition stimulation of EPCs with exogenous s1p peptide significantly decreased apoptosis and increased SDF-1- induced migration. These results were confirmed
In vivo
using
FHL-2
knockout (FHL-2 -/-) mice. Moreover, apoptosis was significantly decreased and migration increased in endothelial cells exposed to the conditioned medium of
FHL-2
-/-
vs. WT EPCs. These effects were abolished by
VPC23019
, an antagonist of sphingosine- 1- phosphate receptor- 1 and 3. Finally, reendothelialization after focal carotid endothelial electric injury in WT mice was significantly increased after application of spleen-derived progenitor cells from
FHL-2
-/-
mice vs. WT mice.

Conclusions:
Our findings suggest that FHL-2 negatively regulates early outgrowth EPC function and secretion of paracrine factors. FHL-2 inhibition reduces apoptosis, enhances survival and migratory capacity of EPCs and ECs by upregulating SK-1/s1p pathway, integrin subunits and cortactin; which results in the improvement of endothelial regeneration.
Abstract 203: Silencing of Macrophage Scavenger Receptor SR-A or CD36 Modulates Transcription Factor NF-κB Activity by Distinct Mechanisms
Authors: Mäkinen Petri I , Lappalainen Jari P , Kivelä Annukka M , Levonen Anna-Liisa , Ylä-Herttuala Seppo
Published: March 2022

Background:
Macrophage scavenger receptors SR-A and CD36 play a key role in the pathogenesis of atherosclerosis by participating in the uptake of modified lipoproteins, further leading to formation of foam cells. We have recently shown that RNAi-mediated silencing of these receptors decreases atherogenesis in hyperlipidemic LDLR
-/-
/ApoB
100/100
mice. Additionally, we discovered that there is a reciprocal regulation mechanism between these two receptors in mouse macrophages. Because there is limited information on the involvement of transcription factor NF-κB to SR-A and CD36 expression, the aim of this study was to investigate the role of NF-κB in the regulation of these receptors in detail.

Methods and Results:
Lentiviral vector mediated silencing of SR-A and CD36 alters the behavior of transcription factor NF-κB in mouse macrophages. After LPS exposure, SR-A silencing enhanced the classical NF-κB activation measured by activation of NF-κB responsive genes such as M-CSF, MCP-1 and ICAM-1. Moreover, CD36 silencing increased the expression of NF-κB subunit p50 with and without LPS stimulation by 2.7-fold (p<0.001) and 1.4-fold (p<0.01), respectively. Consequently, expressions of several p50 homodimer target genes, such as IL-6 and IL-10, were increased when CD36 was silenced. Finally, two putative NF-κB binding sites and increased p50 binding to SR-A promoter were found by EMSA, suggesting that SR-A is a p50 responsive gene, and that p50 is responsible for the reciprocal upregulation of SR-A when CD36 is silenced.

Conclusion:
These results suggest that SR-A and CD36 modulate macrophage functions under inflammatory conditions, and imply that SR-A and CD36 are important signal transducers in inflammatory events involving NF-κB.
Abstract 21: Sortilin Is a Novel Mediator of Cellular Low-Density Lipoprotein Uptake
Authors: Ding Qiurong , Strong Alanna , Rader Daniel J , Cowan Chad A , Musunuru Kiran
Published: March 2022

Using genome-wide association studies and follow-up functional studies, we recently identified the SORT1 gene as a novel regulator of blood low-density-lipoprotein cholesterol (LDL-C) levels. SORT1 encodes sortilin, also known as neurotensin receptor 3, a protein that functions as a multiligand sorting receptor. Sortilin localizes to various intracellular compartments including the Golgi apparatus and the plasma membrane, and it has roles in both endocytosis and intracellular trafficking of other proteins. To test whether sortilin regulates blood LDL-C levels by influencing cellular uptake of LDL particles, we used a lentiviral delivery system to stably overexpress the wild-type protein or either of two trafficking mutants_the first, designated LAYA, traffics to the plasma membrane but cannot undergo endocytosis, and the second, designated STOP, remains trapped in the Golgi apparatus. When overexpressed in cultured human hepatocellular carcinoma cells, we found that wild-type sortilin dramatically increases LDL uptake into the endolysosomal compartment for degradation, and the LAYA mutant robustly traps LDL particles in the plasma membrane, whereas the STOP mutant does not affect LDL uptake. We observed similar effects in skin fibroblasts taken from a patient with familial hypercholesterolemia_and thus are deficient in LDL receptor function_suggesting that sortilin can facilitate LDL uptake independently of the LDL receptor. As a complementary approach, we have generated human SORT1 knockout cells, using the novel TAL effector nuclease (TALEN) technology, to introduce frameshift mutations into the coding sequence and thereby disrupt the SORT1 alleles. Besides providing a proof-of-principle that we can for the first time rapidly and efficiently generate gene knockouts in human cells, the SORT1 knockout cells are allowing us to assess the effects of sortilin deficiency on LDL uptake.
Abstract 211: Sam68 Is a Transcriptional Repressor of Endothelin-Converting Enzyme-1b Gene and Regulates Blood Pressure
Authors: Zhou Junlan , Wu Min , Cheng Min , Boriboun Chan , Biyashev Dauren , Kishore Raj , Qin Gangjian
Published: March 2022

We have recently reported that E2F2, a transcription factor known for its role in cell proliferation and apoptosis, regulates the expression of endothelin-converting enzyme-

1b (ECE-1b) in endothelial cells and contributes to the maintenance of vascular contractility and blood pressure. However, the molecular mechanisms that underlie this novel, cell-cycle independent function of E2F2 remain largely elusive. In this current study, we profiled E2F2 interactome in the nucleus of human umbilical vein endothelial cells by utilizing a proteomic approach. Of 20 E2F2-interacting proteins identified is the Sam68, a classic RNA-binding protein known as a Src kinase substrate. Both endogenous and ectopically expressed Sam68 interact with E2F2 as confirmed by coimmunoprecipitation. Overexpression of Sam68 repressed whereas knockdown of Sam68 increased E2F2-induced ECE-1b promoter activity and mRNA expression. Chromatin immunoprecipitation analyses further confirmed that E2F2 and Sam68 colocalize on ECE-1b promoter, indicating that ECE-1b is a direct transcriptional target of E2F2 and Sam68. In vivo, Sam68 knockout (KO) mice displayed a significantly lowered blood pressure (BP) as compared to WT littermates (tail-cuff measurements of mean BP, KO vs. WT: 104 ± 6 vs. 123 ± 7 mmHg, n=25 males of age 4-6 months, P<0.05). Together, our studies have revealed a previously unknown function of Sam68 as a transcriptional co-repressor of E2F2 in the regulation of ECE-1b expression. Sam68 may link endothelial Src kinase pathways to BP regulation
Abstract 221: Rap1 Regulates Vascular Smooth Muscle Cell Adhesion, Spreading and Proliferation, Migration and Response to Thrombin: Implication for Neointimal Formation
Authors: Huang Jianhua , Pathak Alokkumar , Stouffer George A
Published: March 2022

Background:
α-thrombin is concentrated at sites of vascular injury and contributes to the formation of neointima after vascular injury. α-thrombin stimulates c-jun N-terminal kinase 1(JNK1) activation and proliferative responses in rat aortic smooth muscle cells (RASMC) and these effects are mediated by αvβ3 integrins. Rap1, a small GTPase member of the Ras family, functions in integrin-mediated responses in SMC but what role, if any, that it plays in mediating α-thrombin-induced growth responses is unknown.

Objectives:
In this study, we investigated the effect of Rap1 specific GTPase-activating protein II (Rap1GAPII) overexpression on α-thrombin-induced proliferation and migration in RASMC.

Results:
α-thrombin (2 units/ml) increased formation of activated Rap1 (Rap1-GTP) in quiescent RASMC as determined by GST-fused RalGDS-RBD pull-down assays. Overexpression of Rap1GAPII by an adenovirus-mediated method obliterated endogenous Rap1 activation and abrogated α-thrombin-induced Rap1 activation in RASMC. Overexpression of Rap1GAPII decreased RASMC adhesion and spreading on fibronectin, inhibited the proliferative effects of α-thrombin and reduced migratory responses in a wound assay significantly. Treatment with α-thrombin in RASMC dramatically increased stress fiber formation and focal adhesion formation, and increased phosphorylation of β3 integrins, focal adhesion kinase (FAK), activation of p44/p42 MAP kinase and JNK1 activity in RASMC, and all of these effects were significantly inhibited by overexpression of Rap1GAPII. Three days after balloon injury of rat carotid arteries, a significant increase in Rap1 activation was observed at the site of injury as compared to the contralateral normal arteries.

Conclusions:
Rap1 is activated by α-thrombin and regulates α-thrombin-induced proliferation, migration, signaling pathways in RASMC. Rap1 activation is significantly increased following rat carotid artery balloon injury suggesting it may play a role in neointimal formation.
Abstract 224: Cleavage of DNA Generated During Myocardial Ischemia/Reperfusion Protects the Heart from Injury
Authors: Savchenko Alexander S , De Meyer Simon F , Gallant Maureen E , Wagner Denisa D
Published: March 2022

Introduction:
Neutrophils play a major role in early response to myocardial ischemia/reperfusion (MI/R) injury. During inflammation, neutrophils may form a meshwork of DNA fibers known as neutrophil extracellular traps (NETs). NETs are injurious to tissues and pro-thrombotic. It has been shown that DNase 1, which cleaves DNA, can disassemble NETs-induced thrombi in a flow chamber and reduce incidence of deep vein thrombosis
in vivo
in mice.

Objective and Methods:
To investigate the effect of DNase 1 on MI/R injury, we used a “closed chest” mouse model of acute myocardial infarction for 60 min, followed by 24 h reperfusion. Mice were treated with DNase 1 or vehicle immediately after reperfusion.

Results:
Mice receiving DNase 1 developed a significantly smaller infarcted area compared to mice treated with vehicle (buffer). Immunohistochemical analysis of the myocardium revealed infiltrated neutrophils positive for Gr-1 and citrulinated histones (citH3). Also, extracellular citH3, an element and marker of NETs formation, was observed in an infarcted area mainly in mice treated with vehicle. Of interest, MI/R caused an increase in circulating nucleosomes and DNase 1 treatment revealed a significant decrease in plasma nucleosome levels, as compared with vehicle-treated mice, 24 h after MI/R.

Conclusions:
We show that DNase 1 reduced MI/R infarct size in mice and the lesser injury was confirmed by histological analysis. MI/R resulted in the presence of extracellular citH3, most likely originating from neutrophils, in myocardial sections of vehicle-treated MI/R mice but less so in mice receiving DNase 1. We also observed a decrease in nucleosomes in plasma from MI/R mice treated with DNase 1. Our results support a protective role for DNase 1 in MI/R.
Abstract 226: Platelet Mitochondria Regulate Thrombotic and Inflammatory Function
Authors: Vitseva Olga , Beaulieu Lea M , Koupenova Milka , Tanriverdi Kahraman , Freedman Jane E
Published: March 2022

Introduction:
Cells require energy provided by glycolytic and mitochondrial sources. Obesity, a health issue worldwide, affects mitochondrial function. Platelets possess mitochondria although their role in platelet function is unclear. We assessed the hypothesis that platelet mitochondria contribute to cellular processes and the mitochondrial disorder in obesity may also affect platelet functions.

Methods:
Platelet aggregation and adhesion were assessed to estimate the effects of selective metabolic inhibitors on platelet functions. Mitochondrial ultrastructure was analyzed by transmission electron microscopy in the presence of these inhibitors. C57BL/6J mice were used to investigate the mitochondrial contribution to the effects of high fat diet (HFD) on platelet adhesion. mRNA expression in mouse platelets was measured by RT-PCR.

Results:
The disruption of the mitochondrial function by complementary inhibitors resulted in a reduction of platelet aggregation to 80.9 ± 7.7% (antimycin A), 77.5 ± 9.5% (oligomycin), 59.1 ± 3.2% (carbonyl cyanide 3-chlorophenylhydrazone, CCCP), and 37.2 ± 4.3% (potassium cyanide); n=3,
p
<0.0001. Significant morphological changes in platelet mitochondria, including mitochondrial cristae swelling and accumulation of dense masses in the presence of metabolic inhibitors oligomycin and CCCP were observed. A HFD enhanced thrombin-induced platelet adhesion (44.2 ± 8.7% vs normal chow 30.6 ± 1.3%), which was abrogated in the presence of metabolic inhibitors (19.4 ± 7.1% vs normal chow 14.6 ± 5.4% in the presence of oligomycin; 4.5 ± 2.1% vs normal chow 6.6 ± 2.0% in the presence of CCCP; n=7,
p
<0.001). Mitochondrial, inflammatory, and thrombotic transcripts were all affected in platelets by a HFD. Expression of mitochondrial (mitofusin 2)- and thrombotic (CD41)-related genes were downregulated, while inflammatory-related genes TLR2 and IL1R1 were upregulated in mouse platelets after 3 weeks of a HFD. In conclusion, platelet mitochondria maintain homeostasis and regulate thrombotic and inflammatory function. Mitochondrial dysfunction in obesity affects platelet function, potentially contributing to cardiovascular disease risks associated with the metabolic syndrome.
Abstract 227: Edaravone Injected at the Start of Reperfusion Reduces Muscle Injury Following Leg Ischemia in Rats
Authors: Yamamura Mitsuhiro , Miyamoto Yuji , Mitsuno Masataka , Tanaka Hiroe , Ryomoto Masaaki , Fukui Shinya , Yoshioka Yoshiteru
Published: March 2022

Introduction & Hypothesis:
It is well known that free radicals cause reperfusion injury after leg ischemia. At ATVB 2010 & 2011, we have reported that preoperative injection of the free-radical scavenger, edaravone (Radicut®, Mitsubishi Tanabe Pharma Co., Japan) prevented reperfusion injury. In this study, we evaluated whether edaravone can reduce reperfusion injury if injected at the start of reperfusion.

Methods:
Male Lewis rats (525 ± 78 g, n = 10) were subjected to reperfusion injury models by clamping the bilateral common femoral arteries for 5 hours followed by de-clamping. Rats were divided into two groups and intraperitoneally injected with edaravone (group edaravone; 9.0 mg / kg, n = 5) or saline (control group; n = 5) at the same time as removing the clamps (i.e., the start of reperfusion). Five hours after de-clamping, the muscles of lower extremity were harvested. Tissues were stained with hematoxylin & eosin (HE), in order to count the viable muscle cells. They were also stained with periodic acid-Schiff (PAS), in order to assess the glycogen storage in muscle cells. The number of viable muscle cells and the percentages of PAS-positive area were measured on each three different field (original magnification 200x).

Results:
In the control group, most of the muscle cells were swelling. The density of viable muscle cells in the group edaravone was significantly greater than that of control group (593±60 cells/mm2 vs. 258±31 cells/mm2, p < 0.01). The mean percentage of PAS-positive area in the edaravone group was also significantly higher than that in control group (30.1 ± 6.9 % vs. 7.3 ± 2.1%, p < 0.001, Figure attached).

Conclusions:
Our results suggest that edaravone injected at the start of reperfusion can also reduce muscle injury following leg ischemia in rats, storing a high level of glycogen in viable muscle cells. Therefore, edaravone might be useful in clinical settings following leg ischemia.
Abstract 237: VEGF-Mediated Phosphorylation of eNOS Regulates Angioblast and Endothelial Cell Proliferation
Authors: Gentile Carmine , Muise-Helmericks Robin , Drake Christopher J
Published: March 2022

To evaluate when nitric oxide (NO) is first expressed in the EC lineage, the expression pattern of eNOS, phosphorylated eNOS (P-eNOS), and key proteins that define the endothelial lineage (i.e., Flk-1, TAL-1, CD31) were assessed in 7.0-8.5dpc mouse embryos. Immunohistochemical analyses revealed that embryonic endothelial cells (Flk-1
+
/TAL-1
+
/

CD31
+

) expressed eNOS prior to their investment by smooth muscle cells while isolated angioblasts (Flk-1
+
/TAL-1
+
/

CD31
-

) did not express eNOS. Based on eNOS expression we identified a cell type, transitional angioblasts (

eNOS
+
/

FLK-1
+
/TAL-1
+
/

CD31
-

), intermediate between embryonic endothelial cells (EECs) and angioblasts. Transitional angioblasts are further distinguished from angioblasts by their initiation of cell-cell contacts with other eNOS
+
Flk-1
+
/TAL-1
+
/CD31
-
cells or with EECs. Analysis of P-eNOS and phospho-histone H3 expression in transitional angioblasts and EECs showed a tight correlation between P-eNOS expression and cell proliferation. This correlation was also observed in cultured human umbilical vein endothelial cells (HUVECs) treated with nocodazole to induce a G2/M cell cycle block. Given that VEGF mediates both EC division and NO production, we evaluated whether VEGF-mediated NO production is critical for neovascular processes (vasculogenesis/angiogenesis). We show using allantoic cultures that VEGF-mediated mitosis in Flk-1
+
cells is decreased by the eNOS inhibitors L-NIO and 17-AAG, suggesting that VEGF-mediated eNOS phosphorylation is regulating cell proliferation. Supporting such a role, we found that the alterations in VEGF-mediated vascular patterning we observed in response to eNOS inhibitors were consistent with an overall reduction in the EEC numbers and that VEGF-induced NO production in HUVECs increased the S-nitrosylation of proteins important for the regulation of the G2/M transition, such as cyclin B. Taken together, our findings suggest that VEGF-mediated eNOS phosphorylation regulates transitional angioblast, EEC, and EC proliferation.
Abstract 238: Angiogenesis Requires G-Protein-Coupled Receptor Kinase 2-Interacting Protein 1 (GIT1): A Novel GIT1-ERK1/2-Cortactin Pathway Controls Endothelial Cell Directional Migration
Authors: Majumder Syamantak , Pang Jinjiang , Yin Guoying , Berk Bradford C
Published: March 2022

Introduction—
GIT1 is a multidomain scaffold protein involved in diverse cellular processes including cell adhesion and migration. GIT1 is required for vascular endothelial cell growth factor (VEGF) signaling in endothelial cells (EC) and translocates to lamellipodia during directional migration. Lamellipodia are regions of very rapid actin remodeling which is mediated in part by the cortical actin remodeling protein, cortactin. We hypothesize that GIT1 mediates directional migration of EC by regulating cortactin localization and activation.

Methods and Results—
To determine the role of GIT1 in directional migration of EC, we used a Dunn’s chamber and in vitro scratch wound healing assay. In Dunn’s chamber, the migration of GIT1 siRNA transfected Human Umbilical Vein EC (HUVEC) was decreased by 66±9.1% compared to control siRNA transfected EC in response to 50ng/ml VEGF gradient. Furthermore, the extent of wound healing was decreased by 63±7.8% in GIT1 knock out (GIT1-KO) mouse lung microvascular EC (MLMEC). We next studied the localization pattern of cortactin in MLMEC from GIT1 wild type (GIT1-WT) and GIT1-KO mice. In GIT1-KO MLMEC the number of cells with cortactin localized by immunofluorescence (IF) at the leading edge during wound healing was reduced by 73±5.7% compared to GIT1-WT. Dual IF for cortactin and GIT1 at the leading edge of HUVEC showed extensive co-localization. In response to 50ng/ml VEGF for 15 min, serum deprived HUVEC exhibited a 75.6±11.3% increase in GIT1-cortactin association as demonstrated by co-immunoprecipitation (IP). Among GIT1 domains, the Spa homology domain (SHD) is unique to GIT1 and is required for signal transduction. We next transfected GIT1-KO MLMEC with GIT1(1-770), GIT1(1-420) that contains SHD, and GIT1(1-250+420-770) that lacks SHD. IF and IP analysis of MLMEC showed that SHD was required for cortactin membrane localization and cortactin-GIT1 association. Finally, we showed that GIT1 regulates VEGF induced ERK1/2 mediated phosphorylation of cortactin Ser405, a requirement for cortactin activation.

Conclusion—
The unique SHD of GIT1 is essential for VEGF induced EC migration by assembling a GIT1-ERK1/2-cortactin complex suggesting that SHD of GIT1 may be a therapeutic target to limit pathological angiogenesis.
Abstract 248: TGF-β Neutralization Attenuates AngII-Induced Ascending Aortic Aneurysms While Not Affecting Abdominal Aortic Aneurysms
Authors: Chen Xiaofeng , Rateri Debra L , Howatt Deborah A , Balakrishnan Anju , Moorleghen Jessica J , Daugherty Alan
Published: March 2022

Introduction and Objectives:
We have demonstrated previously that angiotensin II (AngII) induces ascending and abdominal aortic aneurysms (AA) in C57BL/6 mice. In an experimental mouse model of Marfan’s Syndrome that expresses Fbn1
C1039G/+
, ascending AAs were prevented by both a neutralizing antibody to transforming growth factor-β (TGF-β) signaling and the Ang II receptor antagonist, losartan. Conversely, it has been recently reported that a neutralizing TGF-β antibody leads to abdominal aortic rupture during AngII infusion. The aim of this study was to determine the effects of a TGF-β neutralizing antibody on AngII-induced ascending and abdominal AA formation in normolipidemic mice.

Methods and Results:
Eight week old, male, C57BL/6 mice were fed a normal laboratory diet and infused subcutaneously with AngII (1,000 ng/kg/min) by osmotic mini-pumps for 28 days. Mice were injected intraperitoneally with control IgG (R&D Systems, AB-105-C, 10 mg/kg twice a week), or TGF-β IgG antibody (R&D Systems, AB-100-NA, 10 mg/kg per week or twice a week; n=10 mice per group). There were no changes in body weight among the groups. As expected, both doses of TGF-β antibody significantly decreased serum TGF-β concentration (P<0.05). Ascending aortic area was significantly reduced in mice administered with TGF-β antibody at 10 mg/kg twice per week, compared to mice injected with control IgG (P=0.002). There was no difference of incidence of abdominal AA among the control and TGF-β IgG antibody groups.

Conclusion:
In agreement with the effects of TGF-β neutralization in the Fbn1
C1039G/+
transgenic mouse, ascending AA were attenuated by the TGF-β neutralizing antibody during AngII infusion. However, we were unable to replicate the recent finding that TGF-β neutralization leads to increased rupture of abdominal aortas during AngII infusion.
Abstract 26: Novel Role of Anticoagulant Heparan Sulfate in Antithrombin Anti-inflammatory Signaling
Authors: Smits Nicole C , Shworak Nicholas W
Published: March 2022

Antithrombin (AT) therapy reduces morbidity/mortality in systemic inflammatory response syndromes, such as septic shock. These effects stem from the ability of AT to directly initiate cell signaling, which requires AT engagement with heparan sulfate proteoglycans. It is presently unclear if this process involves anticoagulant heparan sulfate (HS
AT+
), a specific motif with high affinity for AT that is produced by endothelial cells. If HS
AT+
regulates anti-inflammatory signaling, then

Hs3st1
-/-

mice, which lack endothelial HS
AT+
, should exhibit a proinflammatory state and should not benefit from AT treatment. We tested these hypotheses using a mouse model of lipopolysaccharide (LPS)-induced septic shock. Hemodynamics were monitored with a left ventricle pressure-volume catheter. Both
Hs3st1
+/+
and
Hs3st1
-/-
mice die of cardiogenic shock. However,
Hs3st1
-/-
, compared to
Hs3st1
+/+
, mice exhibited a much more rapid onset of death. Left ventricular gene expression profiles of mice with systemic LPS challenge showed enhanced expression of leukocyte recruitment genes (P-selectin, Cxcl10) in
Hs3st1
-/-
mice. Consistent with these data, LPS-treated
Hs3st1
-/-
mice, compared to
Hs3st1
+/+
, exhibited enhanced leukocyte rolling flux within cremaster muscle venules. Thus, mice lacking HS
AT+
exhibit a proinflammatory phenotype. AT treatment of
Hs3st1
+/+
mice reduced both LPS-induced lethality and leukocyte-endothelial interactions. Conversely AT treatment of
Hs3st1
-/-
mice enhanced both LPS-induced lethality and leukocyte-endothelial interactions. Thus, AT failed to elicit anti-inflammatory effects in mice lacking HS
AT+
. Our results indicate that endothelial HS
AT+
is one component that mediates AT’s anti-inflammatory activity. These findings provide an initial step towards elucidating the role of HS
AT+
and AT in a novel natural anti-inflammatory pathway of the blood vessel wall. This pathway is an ideal target for developing therapeutics to combat the devastating outcomes of septic shock.
Abstract 261: Differential Regulation of Apolipoprotein E Secretion from Primary Human Macrophages by Different Protein Kinase C Isoforms
Authors: Karunakaran Denuja , Kockx Maaike , Owen Dylan , Jessup Wendy , Kritharides Leonard
Published: March 2022

Macrophage-specific apolipoprotein E (apoE) secretion plays an important role in atherosclerosis. Recently, we established that inhibition of protein kinase A, phospholipase C, protein phosphatase 2B and calcium results in a reduction in apoE secretion. Research suggests PKC may have a role in this process, but the isoforms and pathways involved in human macrophages are unknown. Here, we investigate a role for PKC in regulating apoE secretion from primary human monocyte-derived macrophages (HMDM). HMDM treated with pan-PKC inhibitors (Calphostin C, Ro-318220, Bisindolylmaleimide I) that primarily target the classical and novel PKC isoforms result in a dose-dependent decrease in apoE secretion. Further, the specific classical PKC inhibitor, Gö6976, also resulted in a reduction in apoE secretion. Metabolic labelling studies indicate that CalpC and Gö6976 have no effect on apoE synthesis or degradation but directly inhibit the secretion of apoE. This occurred post-golgi as evidenced by normal glycosylation of apoE on 2D electrophoresis. Direct visualisation of apoE-GFP containing vesicles in HMDM by live cell confocal microscopy demonstrated a marked reduction in vesicular speed from 0.42μm/s in control cells to 0.14μm/s or 0.15μm/s by CalpC and Ro-318220 respectively. Finally, siRNA knockdown (∼70%) of the classical PKC isoforms, PKCalpha and PKCbeta, resulted in decreased basal apoE secretion (∼50%), whereas knockdown of PKCdelta (novel PKC isoform) did not. Interestingly, preliminary data demonstrate that treatment of HMDM with the PKCtheta or PKCzeta pseudosubstrate inhibitor increases apoE secretion from HMDM, whereas both the PKCepsilon translocation inhibitory peptide and siRNA knockdown of PKCepsilon did not affect apoE secretion. This suggests that apoE secretion is differentially regulated by different and specific PKC isoforms. Inhibition of PKCalpha and/or PKCbeta decreases apoE secretion by targeting the apoE trafficking pathway in a post-golgi vesicular compartment, whereas inhibition of PKCzeta (atypical PKC isoform) or PKCtheta (novel PKC isoform) increases apoE secretion. Future studies will further confirm and investigate the role of these novel and atypical PKC isoforms in regulating apoE secretion.
Abstract 268: Resolvin D1 Inhibits Neointimal Formation After Vascular Injury in Mice
Authors: Haller Carolyn A , Xiao Jiantao , Chaikof Elliot L
Published: March 2022

Background:
Resolvin D1 (RvD1), a docosahexaenoic acid (DHA) - derived lipid mediator generated during the resolution of inflammation, possesses potent anti-inflammatory properties. The present study uses a well defined carotid ligation model to investigate the role of RvD1 in the attenuation of neointimal hyperplasia.

Methods:
Left common carotid artery ligation was performed in C57BL/6 mice, RvD1 (10 ng/day) or vehicle control was delivered intravenously for 8 consecutive days. Standard morphometric analysis was performed at 28 days post-ligation. Immunohistochemical characterization of leukocyte infiltration and biochemical characterization of tissue reactive oxygen species were performed at 1, 4, 7, 10 days post-ligation. Chemotaxis experiments were performed using isolated human neutrophils, human coronary artery smooth muscle cells (SMC), and murine thoracic aorta SMC in a standard boyden chamber assay.

Results:
RvD1 treatment afforded an early dampened inflammatory response with reduced infiltration of neutrophils and macrophages and lower detectable levels of tissue myeloperoxidase and superoxide within the first 7 days post-ligation. Morphometric analysis at 28 days post-ligation confirmed a markedly attenuated intimal area (8.37 vs. 33.06,
P
< 0.01) and intimal to media ratio (0.23 vs. 0.83,
P
< 0.01) in RvD1 animals when compared to control. In addition, intimal SMCs were significantly reduced in RvD1 treatment vs. control (30.91 vs. 41.02,
P
< 0.01). In vitro chemotaxis experiments indicated RvD1 can significantly inhibit both neutrophil (1.39 vs. 3.6,
P
< 0.05) and SMC (1.67 vs. 2.32,
P
< 0.05) migration index to inflammatory stimulus leukotriene B4.

Conclusions:
Our results identify the therapeutic potential of RvD1 to attenuate the early inflammatory response, smooth muscle cell migration, and subsequent hyperplasia associated with pathological remodeling following vascular wall insult.
Abstract 278: Signaling Pathways Involved in the Cytoprotective Effects of Erythropoietin and Erythropoietin Derivatives in Ischemic Human Myotubes
Authors: Yu Rebekah Sian Hwee , Abraham David , Hamilton George , Baker Daryll , Tsui Janice
Published: March 2022

Objectives:
Erythropoietin (Epo) has tissue-protective effects in response to injury, acting through the EpoR-βcR heteroreceptor. We have previously shown presence of the EpoR in human skeletal muscle. Here we aim to show the direct interaction of the EpoR with the βcR. Further, we investigate the potential cytoprotective effects of Epo and an Epo-derivative (ARA-290) in a human
in vitro
model of skeletal muscle and establish the role of PI3K/Akt pathway in protecting cells from apoptosis.

Methods:
Ethical approval and informed consent was obtained for gastrocnemius biopsies. Immunohistochemistry and western blot analysis were performed to demonstrate expression of EpoR and βcR in human skeletal muscle. Co-immunoprecipitation (Co-IP) was performed to demonstrate heterodimerisation of the EpoR with βcR. Human myoblasts were isolated from muscle biopsies to investigate the cytoprotective effects of Epo and ARA-290 on myotubes subjected to simulated ischaemia. The PI3k inhibitor, wortmannin, was then used to determine the role of PI3k/Akt pathway in mediating cytoprotection. Western blot analysis, using the pro-apoptotic marker cleaved caspase-3 was performed and compared with levels of Akt and phosphorylated-Akt, using western blot analysis.

Results:
The presence of both EpoR and βcR in human skeletal muscle has previously been demonstrated. We have now confirmed heterodimerisation of EpoR-βcR complex by Co-IP. Exogenous administration of Epo and ARA-290 were able to ameliorate the ischaemia-induced apoptosis on isolated human myotubes as shown by a significant reduction in cleaved caspase-3 expression. Addition of wortmannin, to ARA-290 or Epo pre-treated cells, abolished the reduction in apoptosis. Further, a reduction in apoptosis was associated with an increase in phosphorylated-Akt on western blot analysis.

Conclusion:
We have demonstrated EpoR-βcR heteroreceptor in human skeletal muscle. The ability of ARA-290 to attenuate apoptosis in human myotubes undergoing ischaemic insult suggests a potential role in tissue protection in skeletal muscle injury. We propose that the PI3k/Akt signalling pathway is involved in mediating this cytoprotection.
Abstract 288: An Improved Method for the Isolation of HDL from Human Serum
Authors: Collins Heidi L , Sulpizio Anthony C , Adelman Steven J
Published: March 2022

In an effort to reduce the time and labor resources required for the standard HDL isolation protocol using sequential ultracentrifugation, our laboratory has investigated a modified process for isolation of HDL from human serum. Our method employs an alternative step for removing VLDL and LDL from human serum that requires less than one hour and replaces the first 18 hour ultracentrifugation step. This alternative process is based on the well documented ability of polyethylene glycol (PEG) to selectively and irreversibly precipitate apolipoprotein B-containing lipoproteins. Subsequently, both methods then use a 21 hour ultracentrifugation spin to separate HDL from all other non-lipoprotein components of human serum. For this study comparing the methods, 1.5 ml of human serum was used. The human serum starting material had a total cholesterol value of 262 mg/dL with the following lipoprotein cholesterol profile: 178 mg/dL LDL-C (68%), 34 mg/dL VLDL-C (13%) and HDL-C 48 mg/dL (18%). Comparison of the two methods demonstrates similar recovery of HDL-C from human serum, 35% vs 37% for the standard method versus modified method, respectively. The total cholesterol recovered using the standard protocol was slightly higher than the modified method. However, as shown by lipoprotein gel electrophoresis, HDL isolated by the standard method was only 80% pure, with 20% of the cholesterol present as VLDL and LDL contaminants remaining in the preparation. In contrast, using our modified process, isolated HDL was >99% pure with no detectable VLDL or LDL cholesterol present. In summary, our laboratory has developed a methodology for the isolation of HDL from human serum that: 1) requires substantially less time and resources, 2) provides the same recovery of HDL-C from serum as the standard method and 3) provides a higher purity HDL than is achieved from the standard sequential ultracentrifugation method.
Abstract 30: Reciprocal Expression of MRTF-A and Myocardin Mediated by miR-1 Is Crucial for Phenotypic Modulation of Vascular Smooth Muscle Cells
Authors: Minami Takeya , Kuwahara Koichiro , Nakagawa Yasuaki , Takaoka Minoru , Kinoshita Hideyuki , Nakao Kazuhiro , Kuwabara Yoshihiro , Yamada Yuko , Yamada Chinatsu , Shibata Junko , Usami Satoru , Yasuno Shinji , Nishikimi Toshio , Ueshima Kenji , Sata Masataka , Nakano Hiroyasu , Seno Takahiro , Kawahito Yutaka , Sobue Kenji , Kimura Akinori , Nagai Ryozo , Nakao Kazuwa
Published: March 2022

Myocardin-related transcription factor (MRTF)-A is a Rho signaling-responsive co-activator of serum response factor (SRF). Here we show that induction of MRTF-A expression is key to pathological vascular remodeling in mouse models of vascular disease. MRTF-A expression was significantly higher in the wire-injured femoral arteries of wild-type mice and in the atherosclerotic aortic tissues of ApoE
-/-
mice than in healthy control tissues, whereas myocardin expression was significantly lower. In addition, neointima formation in wire-injured femoral arteries in MRTF-A knockout (

Mkl1
-/-

) mice and atherosclerotic lesions in

Mkl1
-/-
;ApoE
-/-

mice were both significantly attenuated. Expression of vinculin, MMP-9 and integrin β1, three SRF targets and key regulators of cell migration, in injured arteries was significantly weaker in

Mkl1
-/-

mice than in wild-type mice. In cultured vascular smooth muscle cells (VSMCs), a cellular model of dedifferentiated VSMCs, knocking down MRTF-A reduced expression of these genes and significantly impaired cell migration. Underlying the increased MRTF-A expression in dedifferentiated VSMCs was the downregulation of microRNA-1 concomitant with a decrease in myocardin expression. Moreover, the MRTF-A inhibitor CCG1423 significantly reduced neointima formation following wire injury in mice. MRTF-A could thus be a novel therapeutic target for the treatment of vascular diseases.
Abstract 301: MK-886 (5-Lipoxygenase Inhibitor) and DITPA (Thyroid Hormone Analog) Attenuate Global Myocardial Ischemia Reperfusion Injury After Heterotopic Heart Transplantation in Male Rats
Authors: Alamran Fadhil
Published: March 2022

Background:
Heart transplantation is a widely accepted therapy for most patients under 65 years of age with advanced heart failure who remain symptomatic with the expectation of high intermediate term mortality, despite optimal heart failure medications. Global myocardial ischemia reperfusion injury after heart transplantation is believed to impair graft function and aggravate both acute and chronic rejection episodes.

Objective:
this study was undertaken to assess the possible protective effect of MK-886 (5-lipoxigenase inhibitor) and 3,5-Diiodothyropropionic Acid (DITPA) (thyroid hormone analog) against global myocardial ischemia reperfusion injury after heart transplantation by (using heterotopic heart transplantation model ) via interfering with inflammatory pathways.

Materials and Methods:
adult albino rats were randomized into 4 groups (12 rats in each group, 6 donors and 6 recipients ) as follow: group I (N=6), sham group, rats underwent the same anesthetic and surgical procedures (for an identical period of time for global myocardial ischemia and reperfusion through transplantation ) but with no heterotopic heart transplantation; group II, control group, rats underwent 30 min of global myocardial ischemia followed by 60 min of reperfusion via heterotopic heart transplantation; group III, MK-886 treated group, Donors and recipients rats received MK-886 (0.6 mg/kg) i.p. injection 30 min before transplantation, and the same dose was repeated for recipients upon reperfusion; group IV, DITPA treated group, Donors and recipients rats pretreated with DITPA(3.75 mg/kg) s.c. for 7days before transplantation.

Results:
compared with sham group, the levels of cardiac TNF-α, IL-1β, ICAM-1 and plasma level of cardiac troponin I (cTnI) were increased in control group (
p
<0.05). Histologically all rats in control group showed significant cardiac injury (
p
<0.05).

Both MK-886 and DITPA significantly counteract the increase in the levels of cardiac TNF-α, IL-1β, ICAM-1 and plasma level of (cTnI) (
p
<0.05). Morphologic analysis showed that both MK-886 and DITPA markedly improved (
p
<0.05) the severity of cardiac injury in the heterotopically transplanted rats.

Conclusions:
the results of this study reveal that both MK-886 and DITPA may ameliorate global ischemia reperfusion injury after heart transplantation via interfering with inflammatory pathway.
Abstract 304: Mutational Analysis of M-T7, a Viral Anti-inflammatory Protein, Provides Insight into Its Activity
Authors: Bartee Mee , Chen Hao , Davids Jennifer , Saikaly Sami , Lucas Alexandra
Published: March 2022

Viruses express an arsenal of anti-inflammatory proteins that are being developed as immunotherapeutic drugs to treat cardiovascular disease. M-T7, a myxoma viral protein, has been shown to significantly block cell invasion and plaque growth after balloon angioplasty injury and transplantation. The binding target of M-T7 during a myxoma infection is IFNγ, however
in vitro
, M-T7 has been shown to bind a broad class of chemokines, C, CC, and CXC. To understand the mechanism by which M-T7 disrupts chemokine interactions and inflammatory cell responses, M-T7 mutants were generated and characterized
in vitro
for changes in cell activation and migration by the chemokine RANTES. Experiments with a balloon angioplasty model monitored changes
in vivo
. M-T7 mutants were generated to characterize the interactions with chemokines. Protein was expressed in a baculovirus mediated expression system and purified with a Cobalt affinity column. Changes in membrane fluidity were measured with BPP after RANTES activation. As expected, wildtype M-T7 decreased the I
ex
/I
mon
fluorescence ratio when compared to RANTES activated saline controls. However, mutants F137D, R171E, and E209I did not alter membrane fluidity. Interestingly, when cells were activated with PMA, F137D had a decreased I
ex
/I
mon
ratio (P<0.04). M-T7 and mutants decreased cell migration of THP-1 cells (P<0.001) in an in vitro cell migration assay using RANTES as the chemoattractant. Treatment with heparin increased cell migration of M-T7 (P<0.001) and R171E (P<0.029) treated cells, suggesting inhibition of RANTES binding by heparin, but no change for F137D and E209I. With PMA activated cells, migration was disrupted with R171E and E209I (P<0.001) treatment, but not with M-T7 and F137D. Balloon angioplasty was performed on ApoE
-/-
mice, and treated with 100ng/g of M-T7/mutant. Change in the vascular architecture was monitored at the site of injury by measuring plaque size and the Internal Elastic Lamina. F137D and wildtype decreased plaque, but R171E treated mice had significantly increased plaque (P<0.018) and increased IEL (P<0.024). E209I treated mice trended toward an increase in plaque and IEL size.

Of the three mutants, F137D was the most effective in vivo. It differed from the other mutants by reducing the I
ex
/I
mon
ratio of PMA activated cells and binding to RANTES was also inhibited when treated with heparin suggesting these two characteristics are important for its in vivo activity. In contrast, R171E, which does not reduce plaque formation, does not reduce PMA induced cell activation and activity is not altered by the addition of heparin.